Long-term effects of aerobic plus resistance training on the adipokines and neuropeptides in...

Long-term effects of aerobic plus resistance training on theadipokines and neuropeptides in nonalcoholic fatty liverdisease obese adolescentsAline de Pianoa, Marco T. de Mellob, Priscila de L. Sanchesa,Patrıcia L. da Silvaa, Raquel M.S. Camposa, June Carniera,Flavia Corgosinhoa, Denis Foschinia, Deborah L. Masquioc, Lian Tocka,Lila M. Oyamaa, Claudia Maria da Penha Oller do Nascimentoa,Sergio Tufikb and Ana R. Damasoa,c,d

Objective To compare the effects of aerobic training

(AT) with aerobic plus resistance training (AT + RT)

in nonalcoholic fatty liver disease (NAFLD) obese

adolescents.

Design Long-term interdisciplinary weight-loss therapy

(1 year of clinical, nutritional, psychological, and exercise-

related intervention).

Participants Fifty-eight postpubertal obese adolescents

were randomized to AT or AT + RT according to NAFLD

diagnosis. Adipokine and neuropeptide concentrations

were measured by enzyme-linked immunosorbent assay,

visceral fat by ultrasound, and body composition by

plethysmography.

Results The NAFLD group that followed the AT + RT

protocol presented lower insulin, homeostasis model

assessment-insulin resistance (HOMA-IR), and alanine

transaminase (ALT) values after intervention compared

with AT. It was verified that there was a higher magnitude of

change in the subcutaneous fat, glycemia, total cholesterol

(TC), low-density lipoprotein-cholesterol, ALT, and

adiponectin in response to AT + RT than in the control

group (AT). All patients who underwent the AT + RT

exhibited significantly higher adiponectin, leptin, and

Dadiponectin and lower melanin-concentrating hormone

(MCH) concentrations after therapy compared with the

AT group. In the simple linear regression analysis, changes

in glycemia, insulin, and HOMA-IR were independent

predictors of significant improvement in adiponectin

concentration. Indeed, DAST (aspartate transaminase)

and DGGT (c-glutamyl transpeptidase) were independent

predictors of DALT, while Dfat mass and DAgRP

(agouti-related protein) were independent predictors of

DMCH. Although the number of patients was limited, we

showed for the first time the positive effects of AT + RT

protocol in a long-term interdisciplinary therapy to improve

inflammatory biomarkers and to reduce orexigenic

neuropeptide concentrations in NAFLD obese adolescents.

Conclusion The long-term interdisciplinary therapy

with AT + RT protocol was more effective in significantly

improving noninvasive biomarkers of NAFLD that are

associated with the highest risk of disease progression

in the pediatric population. Eur J Gastroenterol Hepatol

24:1313–1324 �c 2012 Wolters Kluwer Health | Lippincott

Williams & Wilkins.

European Journal of Gastroenterology & Hepatology 2012, 24:1313–1324

Keywords: adolescent, exercise, nonalcoholic fatty liver disease, obesity

aPost Graduate Program of Nutrition, bPsycobiology Department, cPost GraduateProgram of Interdisiciplinary Health Sciences and dBiosciences Department,Federal University of Sao Paulo, Paulista Medicine School, UNIFESP-EPM,Sao Paulo, Brazil

Correspondence to Ana R. Damaso, PhD, Street Professor Francisco de Castro,No. 93, Vila Clementino, Sao Paulo, SP 04020 050, BrazilTel/fax: + 55 11 5572 0177;e-mail: [email protected], [email protected]

Received 25 April 2012 Accepted 27 June 2012

IntroductionDuring recent decades exercise has emerged as a key

adjuvant tool in the control of many diseases, including

dyslipidemia, hypertension, diabetes, cancer, asthma,

obesity, metabolic syndrome, and also recently nonalco-

holic fatty liver diseases (NAFLD) [1–3].

Moreover, it was recognized by the American College of

Sports Medicine (ACSM) that ‘exercise is medicine’ in

view of reinforcing the important role of this approach in

overall lifestyle intervention around the world. Since then

there has been a consensus that applying some types of

exercise is desirable as adjuvant strategy in clinical

council in a constellation of chronic diseases [4].

As previously demonstrated by our research group and

others, in the literature many systems in the body present

function changes that induce the coexistence of two

or more diseases, including pediatric obesity and

NAFLD [5,6]. In fact, it was shown that B50% of obese

adolescents had some degree of NAFLD, and it was

demonstrated that an interdisciplinary therapy including

Original article 1313

0954-691X �c 2012 Wolters Kluwer Health | Lippincott Williams & Wilkins DOI: 10.1097/MEG.0b013e32835793ac

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

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clinical and behavioral modifications, and nutritional and

aerobic exercise was effective in reducing this prevalence

and in improving some liver metabolic functions [7].

Adipokine imbalance has been observed in both adults

and children with nonalcoholic steatohepatitis (NASH).

Adipokines exert an important role in liver disorders,

leading to hepatocellular damage, inflammation, fibrosis,

and progressive liver disease [8,9].

Adipokines produced partially by inflammatory cells

infiltrating adipose tissue have a key function in the

pathogenesis of insulin resistance and NAFLD, through

complex and interactive paracrine and endocrine me-

chanisms [10]. In obesity, the adiponectin and leptin

presented dysregulation. Leptin concentrations rise, but

tissue leptin develops, thereby impairing the ability of

leptin to decrease food intake, increase energy expendi-

ture, and prevent partitioning of lipid into ectopic stores

such as muscle and liver.

In contrast, adiponectin concentrations are decreased in

patients with NAFLD, attenuating the anti-inflammatory

and proproliferative effects of this adipokine, altering

lipid partitioning in the hepatocytes, because of the

important role of adiponectin in the inhibition of

lipogenesis, and activating fatty acid oxidation through

effects on AMPK and PPAR-a [11,12]. Serum adiponec-

tin concentration is low in patients with NASH compared

with age/sex/BMI-matched controls; however, it could not

accurately predict NASH versus steatosis alone with

sensitivity and specificity [5].

In agreement, a recent study developed by our group

showed that NAFLD obese adolescents had more altered

neuroendocrine pathways in regulating energy balance and

inflammatory process than the non-NAFLD obese group,

and a negative correlation was observed between neuro-

peptide Y (NPY) and adiponectin concentration [13]. In

addition, saturated fat intake was determined to be a risk

factor for these changes. All together, these results suggest

that NAFLD obese adolescents had impairment in a

weight-loss process [14]. Finally, the important role of a

negative energy balance in using exercise to treat both

obesity and NAFLD needs to be considered in clinical

approaches to promote pediatric health.

In the central nervous system, the arcuate nucleus of the

hypothalamus is crucial for feeding control and contains

two interconnected groups of ‘first-order’ neurons produ-

cing NPY and agouti-related protein (AgRP), both involving

the orexigenic pathways, and pro-opiomelanocortin, co-

caine, and amphetamine-regulated transcript peptide,

which correspond to the anorexigenic pathways [15].

It is suggested that melanin-concentrating hormone

(MCH) acts in concert with other neuropeptides,

including corticotrophin release hormone produced by

neighboring neurons, and NPY or pro-opiomelanocortin

in neurons of the arcuate nucleus, exerting a key role in

triggering food intake [16].

Classically, aerobic exercise is largely recommended and

useful in the control of obesity, in children and

adults [17–19]. Since most patients with NAFLD are obese,

targeting pediatric obesity should help in reducing the

burden of pediatric NAFLD. Therefore, the main target of

NAFLD treatment is to improve quality of life and reduce

long-term cardiovascular and liver morbidity and mortality,

thus reducing the associated comorbidities [5].

Several studies have shown that increased aerobic

exercise on a regular basis improves the metabolic

parameters strongly associated with NAFLD [17–19].

Interestingly, it has recently been shown in obese

adolescents with a diagnosis of metabolic syndrome that

combined exercise [aerobic plus resistance training

(AT + RT)] was more effective than aerobic exercise

alone in improving the related inflammatory process,

including an increase in adiponectin concentration and

control of cardiovascular risk factors [20]. This approach

was performed based on an interdisciplinary therapy as

previously suggested [13,14,21–23]. However, the con-

tribution of AT + RT associated with an interdisciplinary

intervention to key variables of NAFLD development and

to the adipokines and neuropeptides is unclear and

deserves further study. The main aim of the present

research was to compare the effects of two kinds of

exercise in NAFLD obese adolescents, and it was

hypothesized that combined exercise (AT + RT) was

more effective than AT alone in controlling this disease in

obese adolescents. The second aim was to identify

independent predictors of improvement of anti-inflam-

matory adipokine, liver function, and neuropeptides.

Materials and methodsParticipants

A total of 58 obese adolescents (27 boys and 31 girls) who

entered the Interdisciplinary Obesity Program of the

Federal University of Sao Paulo, Paulista Medical School

were assigned to two subgroups: with or without NAFLD.

These patients underwent weight-loss therapy. The

evaluations were performed at baseline and after 1 year

of an interdisciplinary approach.

The age of the participants ranged from 15 to 19 years

(16.48±1.42 years). BMI was 36.55±4.6 kg/m2. All

participants were confirmed as meeting the inclusion

criteria of postpubertal stage V [24] [based on the Tanner

stages of obesity (BMI > 95th percentile of the CDC

reference growth charts)] [25]. Exclusion criteria were

identified as genetic, metabolic or endocrine disease and

previous drug utilization. Informed consent was obtained

from all participants and/or their parents, including

agreement of the adolescents and their families to

participate as volunteers. This study was performed in

accordance with the principles of the Declaration of

1314 European Journal of Gastroenterology & Hepatology 2012, Vol 24 No 11


Helsinki and was formally approved by the institutional

ethics committee (#0135/04) and registered as a clinical

trial (NCT01358773).

Study protocol and medical screening

The participants were medically screened, and their

pubertal stages and anthropometric measures were assessed

(height, weight, BMI, and body composition). The

endocrinologist completed a clinical interview, including

questions to determine eligibility based on inclusion and

exclusion criteria. A blood sample was collected and

analyzed, and ultrasound (US) was performed to measure

visceral and subcutaneous fat and to diagnosis NAFLD.

The patients were then divided into two groups, NAFLD

(n = 28) and non-NAFLD (n = 30), according to US

screening. Subsequently, these two groups were rando-

mized into two subgroups, according to different kinds of

exercise: AT group: 15 patients without NAFLD and 14

with NAFLD and AT + RT group: 15 patients without

NAFLD and 14 with NAFLD (Fig. 1). For all participants,

the procedures were scheduled for the same time of day to

remove any influence of diurnal variations. All participants

underwent an ergometric test. Thereafter, obese adoles-

cents started the interdisciplinary weight-loss program

(described in a later section).

Anthropometric measurements and body composition

Participants were weighed wearing light clothing and no

shoes on a Filizola (Filizola S.A. Pesagem e Automacao, Sao

Paulo, SP, Brazil) scale to the nearest 0.1 kg. Height was

measured to the nearest 0.5 cm by using a wall-mounted

stadiometer (Sanny, model ES 2030; Sao Bernardo do

Campo, Sao Paulo, SP, Brazil). BMI was calculated as body

weight divided by height squared. Body composition was

estimated by plethysmography in the BOD POD body

composition system (version 1.69; Life Measurement

Instruments, Concord, California, USA) [26].

Serum analysis

Blood samples were collected in the outpatient clinic B8 h

after an overnight fast. Insulin resistance was assessed using

the homeostasis model assessment-insulin resistance

(HOMA-IR) index and the quantitative insulin sensitivity

check index (QUICKI). HOMA-IR was calculated using

fasting blood glucose (FBG) and immunoreactive insulin

(I): [FBG (mg/dl)� I (mU/l)]/405 and QUICKI was

calculated as 1/(log I + log FBG). TC, triglycerides (TG),

high-density lipoprotein (HDL), low-density lipoprotein

(LDL), and very-low-density lipoprotein (VLDL) were

analyzed using a commercial kit (CELM, Barueri, Brazil).

The HOMA-IR data were analyzed according to reference

values reported by Schwimmer et al. [27]. The AgRP,

MCH, total adiponectin, and leptin concentrations were

measured using a commercially available enzyme-linked

immunosorbent assay kit (Phoenix Pharmaceuticals Inc.,

Belmont, California, USA) according to the manufacturer’s

instructions.

Visceral and subcutaneous adiposity measurements

All abdominal ultrasonographic procedures and measure-

ments of visceral and subcutaneous fat tissue were

performed by the same physician, who was blinded to

participant assignment groups at baseline and after

intervention. This physician was a specialist in imaging

diagnostics. A 3.5-MHz multifrequency transducer (broad-

band) was used to reduce the risk of misclassification. The

intraexamination coefficient of variation for US was 0.8%.

US measurements of intra-abdominal (visceral) and

subcutaneous fat were obtained. US-determined sub-

cutaneous fat was defined as the distance between the

skin and external face of the rectus abdominis muscle,

and visceral fat was defined as the distance between the

internal face of the same muscle and the anterior wall of

the aorta. Cut-off points to define visceral obesity by

ultrasonographic parameters were based on previous

methodological descriptions by Ribeiro-Filho et al. [28].

Dietary program

Energy intake was set at the levels recommended by the

dietary reference intake for participants with low levels of

physical activity of the same age and sex following a

balanced diet [29]. No drugs or antioxidants were

recommended. Once a week, adolescents had dietetic

lessons (providing information on the food pyramid, diet

record assessment, weight-loss diets and ‘miracle’ diets,

food labels, dietetics, fat-free and low-calorie foods, fats

(kinds, sources and substitutes), fast-food calories and

nutritional composition, good nutritional choices on special

occasions, healthy sandwiches, shakes and products to

promote weight loss, functional foods, and decisions on

food choices). All patients received individual nutritional

consultation during the intervention program.

At the beginning of the study and 12 months into the

program, a 3-day dietary record was collected. Portions

were measured in terms of familiar volumes and sizes.

The dietician taught the parents and the adolescents how

to record food consumption. These dietary data were

transferred to a computer by the same dietician, and the

nutrient composition was analyzed using a software

program developed at the Federal University of Sao

Paulo, Paulista Medical School (Nutwin version 1.5 for

Windows, 2002) that used data from Western and local

food tables. In addition, the parents were encouraged by a

dietician to call if they needed extra information.

Exercise program

Aerobic training

During the 1-year interdisciplinary intervention period, 29

adolescents followed a personalized AT program including a

60-min session three times a week (180 min/week) under

the supervision of an exercise physiologist. Each program

was developed according to the results of an initial oxygen

uptake test for aerobic exercises (cycle-ergometer and

Exercise on the adipokines in NAFLD De Piano et al. 1315


treadmill). The intensity was set at a workload correspond-

ing to a ventilatory threshold of 1 (50–70% of oxygen

uptake test). At the end of 6 months, aerobic tests were

performed to assess physical capacity, and physical training

intensity was adjusted for each individual. During the

aerobic sessions, adolescents underwent heart-rate mon-

itoring. The exercise program was based on the 2001

recommendations provided by the ACSM [30].

Aerobic plus resistance training

During the 1-year interdisciplinary intervention period,

29 adolescents followed the combined exercise training

program. This protocol was performed three times per

week for 1 year and included 30 min of AT plus 30 min of

RT per session. The participants were instructed to

reverse the order of the exercises (aerobic and resistance)

at each training session. The AT consisted of running on a

motor-driven treadmill (model TR 9700HR; Life Fitness,

Schiller Park, Illinois, USA) at a cardiac frequency

intensity representing ventilatory threshold I (±4 bpm),

as determined by the results of an initial oxygen uptake

test for aerobic exercises. The exercise program was

based on guidelines from the ACSM [30].

RT was also designed based on the ACSM recommenda-

tions [30]. Exercises targeted each of the main muscle

Fig. 1

Ethicalcommittee0135/04

Pubertal evaluation

Clinical evaluation ECG

Blood collectbody composition

Diagnoses of NAFLD byultrasonography

30 withoutNAFLD

28 withNAFLD

Psychologicalinterventiononce a week

Nutritionalinterventiononce a week

Exercise interventionthree times/week /1 h

15 patients without NAFLD and14 with NAFLD: aerobictraining (AT group)

15 patients without NAFLDand 14 with NAFLD: aerobicplus resistance training(AT+RT group)

Clinical evaluationonce a week 1 year

Interdisciplinaryintervention

15 to19 years old MediaBMI>95thpercentile

Study protocol diagram. AT, aerobic training; NAFLD, nonalcoholic fatty liver disease; RT, resistance training.



groups. After an introductory period, the load of training

was adjusted such that volume and intensity were

adjusted inversely, decreasing the number of repetitions

to between six and 20 repetitions, for three sets.

Psychological intervention

Diagnoses of common psychological problems associated

with obesity, such as depression, disturbances of body

image, anxiety, and decreased self-esteem, were estab-

lished by validated questionnaires. During the interdisci-

plinary intervention, the adolescents had weekly

psychological support group sessions where they dis-

cussed body image and alimentary disorders, such as

bulimia, anorexia nervosa, and binge eating; their signs,

symptoms and health consequences; the relationship

between their feelings and food; problems in the family,

such as alcoholism; and other topics. Individual psycho-

logical therapy was recommended when we found

individuals with nutritional and behavioral problems.

Statistical analysis

All data were analyzed using Statistica version 6 for

Windows (StatSoft, Tulsa, Oklahoma, USA), with the

significance level set at P value less than 0.05. Data are

expressed as the mean±SD unless otherwise stated.

Distributional assumptions were verified by the

Kolmogorov–Smirnov test, and nonparametric methods

were performed when appropriate. Adipokines and

neuropeptides were analyzed with nonparametric tests

and expressed as median, minimum, and maximum

values. Comparisons between measures at baseline and

after weight-loss intervention were made using an

analysis of variance for repeated measures or the Wilcoxon

signed-rank test of nonparametric variables. Comparisons

between groups were performed using a one-way analysis

of variance or the Mann–Whitney test (nonparametric

variables). Pearson’s correlation was performed to test the

direction and strength of the relationship between

changes in the adiponectin, alanine transaminase

(ALT), and MCH concentrations and the variables of

interest and to select those variables that did not present

collinearity, to select the predictors in the simple

regression. Stepwise simple linear regression analysis

was performed to estimate the association with para-

meters known to influence the improvement of adipo-

nectin, ALT, and MCH concentrations.

ResultsAt the beginning of therapy, 58 obese adolescents were

enrolled in the program. The results are presented for the

whole population studied; we did not find significant sex

differences in BMI at baseline.

Aerobic training group

After weight-loss intervention, we observed significant

improvements in body mass, BMI, and fat mass (kg) in

those patients without NAFLD who underwent the AT.

In addition, the patients with NAFLD presented a

significant reduction in the same variables, as well as in

fat mass (%) and visceral fat (Table 1).

Aerobic plus resistance training group

After weight-loss intervention, the group without

NAFLD that underwent the AT + RT presented sig-

nificant improvement in body mass, BMI, fat mass (kg

and %), glycemia, TC, and LDL-cholesterol (LDL-c).

In patients with NAFLD, we observed a significant

improvement in these same variables, plus a reduction in

the subcutaneous fat (Table 1).

Comparison between the two kinds of exercise

It is important to note that the AT group without

NAFLD presented lower adiponectin and leptin at

baseline compared with the AT + RT group. Meanwhile,

the patients with NAFLD who underwent this same kind

of exercise presented lower adiponectin and MCH

concentrations at baseline. After 1 year of interdisciplin-

ary intervention, the group without NAFLD that

followed the AT + RT protocol presented lower values

of insulin, HOMA-IR, and VLDL-c compared with the

AT group. In addition, this same group presented a higher

magnitude of change (calculated by D) in fat mass (kg and

%), lean mass (kg and %), insulin, HOMA-IR, LDL-c,

adiponectin, adiponectin/leptin ratio, and MCH.

In contrast, the NAFLD group that followed the AT + RT

protocol presented lower values of insulin, HOMA-IR, and

ALT after the long-term intervention compared with AT.

When we analyzed the D after 1 year in these patients, it

was verified that there was a higher magnitude of change in

the subcutaneous fat, glycemia, TC, LDL-c, ALT, and

adiponectin in response to AT + RT.

On the basis of the adipokines and neuropeptides data, it

was verified that all the patients, with and without

NAFLD, who underwent the AT + RT exhibited sig-

nificantly higher adiponectin, leptin, and Dadiponectin,

and lower MCH concentrations after the long-term

therapy compared with the AT group. However, only

the patients without NAFLD who followed the AT + RT

presented a higher magnitude of change in the leptin

concentration (Table 2).

After analyzing the correlation between Dadiponectin and

body composition, lipid and biochemical profile, and

orexigenic neuropeptides, a negative correlation was

found between Dadiponectin and changes in glycemia,

insulin, and HOMA-IR (Fig. 2a–c). Moreover, it was

verified that there was a positive correlation between

change in the orexigenic neuropeptide MCH and fat mass

(kg and %) and AgRP (Fig. 3a–c).

When we performed the simple linear regression analysis of

Dadiponectin, DALT, and DMCH as dependent variables in

obese adolescents adjusted by age and sex, it was revealed



Table 1 Body composition, subcutaneous and visceral adipose tissues, and biochemical parameters in obese adolescents with and without nonalcoholic fatty liver disease according toexercise training

AT (n = 29) AT + RT (n = 29)

Patients without NAFLD (n = 15) Patients with NAFLD (n = 14) Patients without NAFLD (n = 15) Patients with NAFLD (n = 14)

Basal 1 year D1 year Basal 1 year D1 year Basal 1 year D1 year Basal 1 year D1 year

BM (kg) 92.56±10.40 82.96±7.64c – 9.43±8.49 108.85±8.54 97.65±10.75c – 10.86±8.17 99.12±13.40 87.07±9.64c – 12.04±8.49 109.26±9.70 94.81±13.44c – 14.45±7.95BMI

(kg/m2)33.53±3.58 29.95±2.65c – 3.38±2.91 37.99±3.64 34.49±5.13c – 3.72±2.77 36.46±4.49 31.74±3.60c – 4.72±2.97 38.43±5.26 32.98±6.05c – 5.44±2.95

Fat mass(%)

37.36±6.52 33.23±8.01 –7.24±6.02 41.72±5.38 35.40±9.78c – 6.43±6.42 46.24±6.49 35.13±7.76c – 11.11±5.65b 48.92±5.93 40±8.91c – 8.92±4.54

Fat mass(kg)

34.54±6.59 27.40±6.47c – 6.72±7.80 45.44±6.89 35.18±12.22c – 10.24±8.71 44.49±9.41 31.24±9.68c – 14.13±8.56b 53.65±9.36 38.22±11.66c – 15.42±5.73

Lean mass(kg)

58.04±9.33 52.86±9.45 – 2.75±2.48 63.42±7.39 62.45±7.58 – 0.64±3.82 52.82±5.88 56.04±5.76 2.85±2.76b 55.62±6.50 57.76±9.12 1.25±5.87

Visceral(cm)

4.21±1.18 2.82±0.81 – 1.39±1.18 6.31±1.33 3.69±1.37c – 2.61±1.20a 3.77±0.91 1.95±0.53 – 1.81±0.76 4.61±1.16 2.66±0.91 – 1.94±0.82

Sub (cm) 3.69±0.96 3.16±0.85 – 1.09±0.64 3.42±0.87 3.23±1.02 – 0.18±0.62a 3.96±0.53 3.19±0.87 – 0.77±0.99 3.70±0.52c 2.93±0.74c – 0.77±0.76b

Glycemia 90.86±4.71 88.73±6.58 – 2.13±5.76 90.57±4.97 91.84±8.39 1.15±6.80 87.40±5.05 83.60±4.57c – 3.80±6.60 90.42±7.73 85.28±5.36cb – 5.14±6.71b

Insulin 12.2(8.20–21.80)

12.70(3.90–21.30)

– 0.12±5.06 18.95(10.20–63.2)a

15.5 (4.4–43.3) –7.81±17.21 15.6(7.1–23.3)

7.6 (4.1–16.1)b – 5.95±5.11b 20.6 (8.3–31) 10.48(2.9–24.6)b

– 8.87±6.44

HOMA-IR 2.79(1.73–5.00)

2.87(0.77–4.83)

– 0.07±1.23 4.20(2.31–14.46)a

3.47(0.95–10.46)

– 1.63±3.77 3.15(1.45–5.23)

1.49(0.94–3.57)b

– 1.39±1.23b 4.40(1.86–6.63)a

1.63(0.58–5.58)b

– 2.07±1.46

TC (mg/dl) 152.60±26.95 149.06±32.15 – 3.53±15.85 160.71±47.85 161.69±44.27 3.61±23.12 173.66±33.30 156.26±32.26c – 17.40±27.60 167.28±23.71 150.21±21.56c – 17.07±17.70b

LDL (mg/dl) 87±25.03 89.80±28.18 2.80±11.07 90±39.65 92.38±37.57 3.61±16.42 113.13±28.13 96±25.11c – 17.13±19.82b 106.57±22.33 91.28±19.30c – 15.28±13.48b

VLDL 18 (9–42) 10 (6–27) – 6.46±5.33 20.5 (9–50) 20 (5–48)a – 2.92±6.98 16 (12–47) 15 (8–40)b – 1.20±11.45 17.5 (7–41) 15 (6–37) – 1.57±4.95HDL 47.53±9.42 47.66±8.96 0.13±7.20 45.07±9.35 47.23±12.2 2.92±6.19 42.66±5.70 43.60±6.95 0.93±6.32 41.64±8.66 41.42±6.21 – 0.21±5.05TG 89.40±41.82 58.26±27.67 – 31.13±26.28 128.28±66.00 110.38±59.66 – 14.76±34.50 89±43.92 82.66±39.34 – 6.33±56.95 95.28±48.64 87±41.73 – 8.28±25.18AST 22 (15–35) 21 (14–36) – 1.73±4.36 30.5 (17–60)a 25 (20–35) – 6.61±7.65a 22 (16.36) 23 (14–34) – 0.40±5.48 23.50 (18–41) 20.50 (17–53) – 1.57±6.92ALT 30 (19–49) 27 (9–53) – 3.46±10.30 46 (18–146)a 39 (16–77) – 21.84±23.76a 22 (16–31)b 23 (10–46) – 0.06±10.42 23.5 (16–69)b 20 (9–73)b – 5.78±9.73b

GGT 17 (12–33) 16 (5–29) – 2.26±4.57 26.50 (9–155)a 22 (14–47)a – 12.61±29.09 20 (12–57) 20 (11–41) – 3.06±9.62 19 (15–85) 17 (10–65) – 5.35±5.70

ALT, alanine transaminase; AST, aspartate transaminase; AT, aerobic training; BM, body mass; GGT, g-glutamyl transpeptidase; HDL, high-density lipoprotein; HOMA-IR, homeostasis model assessment-insulin resistance; LDL,low-density lipoprotein; NAFLD, nonalcoholic fatty liver disease; RT, resistance training; TC, total cholesterol; TG, triglycerides; VLDL, very-low-density lipoprotein.aGroup with NAFLD versus without NAFLD for the same time (Pr0.05).bGroup aerobic training versus group aerobic plus resistance training for the same time (Pr 0.05).cBasal versus 1 year (Pr0.05).

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that changes in glycemia, insulin, and HOMA-IR were inde-

pendent predictors of a significant increase in adiponectin

concentration. Indeed, DAST (aspartate transaminase)

and DGGT (g-glutamyl transpeptidase) were indepen-

dent predictors of significant changes in ALT, while Dfat

mass (kg and %) and DAgRP were independent predictors

of significance in DMCH (Table 3).

DiscussionThe prevalence of NAFLD in both groups was 48.3% at

the beginning of therapy. After 1 year of the intervention,

there was a significant reduction to 38% in the AT group

and to 17.2% in the AT + RT, showing that this last

exercise protocol was more effective in decreasing the

NAFLD prevalence than AT (data not shown).

In the present investigation, it was shown that both

protocols of exercise in association with an interdisci-

plinary approach were effective in improving metabolic

and hormonal profiles in all analyzed patients (Tables 1

and 2). Overall, these results confirm the importance of

lifestyle changes in ameliorating pediatric health, as

previously demonstrated [1,20,31].

Recent management paradigms are based on the pre-

sence of associated risk factors in a particular patient with

NAFLD, such as obesity and comorbidities [5]. We

showed that patients with NAFLD presented more

altered values of insulin, HOMA-IR, and hepatic

transaminases in the AT group, and it is essential to

treat these alterations, which can be considered impor-

tant risk factors of NAFLD (Table 1). In agreement with

our study, Manco et al. [32], verified that children with

NAFLD presented higher insulin levels and HOMA-IR

than obese controls.

The main goal in the treatment of NAFLD is to improve an

individual’s quality of life and reduce liver morbidity and

mortality, decreasing the associated comorbidities [5], and

our research demonstrated the efficiency of long-term

interdisciplinary intervention, mainly with AT + RT protocol.

The results suggest that both protocols of training

improve more variables in NAFLD obese adolescents,

in addition to fat mass (%), and subcutaneous and visceral

fat, compared with obese without NAFLD. Since the

body fat distribution is a determinant factor in NAFLD

development, it may suggest an important role of both

strategies in its prevention. In fact, previously it was

shown that the expansion of visceral fat is a determining

factor in NAFLD in obese adolescents [33]. However, we

first evidenced that combined training was more effective

than ATalone in promoting a high magnitude of change in

fat mass (kg and %), lean mass (kg and %), insulin,

HOMA-IR, LDL-c, adiponectin, adiponectin/leptin ratio,

and MCH in the group without NAFLD, and major

changes in subcutaneous fat, glycemia, TC, LDL-c, ALT,

and adiponectin in response to AT + RT (Tables 1 and 2).Tab

le2

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A study by Nobili et al. [34] corroborated our data, and

demonstrated that lifestyle intervention consisting of a

diet tailored to the individual’s requirements and

increased physical activity significantly improve the

insulin resistance, serum levels of aminotransferase, lipid

levels, and liver injury of children and adolescents with

NAFLD. A pediatric open-label study showed that a

mean weight loss of B5 kg resulted in improvement in

serum ALT and AST in most children with NAFLD [35].

The pathophysiological basis for the management of

NAFLD is that weight reduction results in white adipose

tissue loss, which decreases insulin resistance. Exercise

can also improve muscular insulin sensitivity, and may

reduce insulin resistance in NAFLD [36].

An association was demonstrated between physical

activity intensity and histological severity of NAFLD.

The results supported an association of vigorous but not

moderate or total exercise with the severity of NAFLD.

Optimal doses of exercise by duration and intensity for

the prevention or treatment of NASH have not been

established; however, intensity may be more important

than duration or total volume [37].

Aerobic exercise training has been shown to reduce

intrahepatic TG and visceral fat even in the absence of

significant weight changes [17]. However, Devries

et al. [2] verified that short-term endurance training

without weight loss did not alter hepatic lipid content.

This result reinforces the importance of long-term

interdisciplinary therapy in the treatment of NAFLD.

Moreover, a combination of diet and exercise appears to

be superior to either diet or exercise alone, promoting

greater improvement in the metabolic profile [18]. In a

study with elderly obese patients, the effects of diet and

diet plus exercise on intrahepatic fat content were

Fig. 2

15r = −0.38; P = 0.07 r = −0.44; P = 0.02(a) (b)

r = −0.48; P = 0.01(c)

25

20

15

10

5

0

−5

−10

−15

−20

−25−6 −4 −2 0 2 4 6 8

10

5

0

−5

−10

−15

−20

−25−6 −4 −2 0

6

4

2

0

−2

−4

−6−6 −4 −2 0 2 4 6 8

ΔAdiponectin

ΔGly

cem

ia

ΔAdiponectin

ΔAdiponectin

ΔIns

ulin

ΔHO

MA

-IR

2 4 6 895% confidenceinterval

95% confidenceinterval


Negative correlation between Dadiponectin and Dglycemia (a), Dadiponectin and Dinsulin (b), and Dadiponectin and DHOMA-IR (c) in the obeseadolescents. HOMA-IR, homeostasis model assessment-insulin resistance.



evaluated. The authors observed that both groups

reduced intrahepatic fat; however, the addition of

exercise to diet therapy improves physical function and

other obesity-related and aging-related metabolic ab-

normalities, such as TG and LDL-c [19].

Mechanisms related to better results of AT + RT can be

hypothesized, such as the maintenance of lean mass that is

considered a powerful tissue metabolically active, favoring

the weight loss process. The most important mechanisms

that can partially explain the greater effect of AT + RT in

NAFLD control is that the contraction of esqueletical

muscle can stimulate GLUT-4 translocation in the absence

of insulin by activating the AMPK enzyme [37].

Thus, the effects of insulin and muscle contraction are

additives, suggesting that insulin and exercise activate

Fig. 3

10

5

0

−5

−10

−15

−20

−25

−30

−35−15 −10 −5 0 5 10 15 20 25


30

10

5

0

−5

−10

−20

−15

−25−15 −10 −5 0 5 10 15 20 25 30

−15−0.4

−0.2

0.0

0.2

0.4

0.6

0.8

−10 −5 0 5 10 15 20 25 30

ΔFat

mas

s (k

g)

ΔFat

mas

s (%

)

ΔAgR

P

ΔMCH

r = 0.38; P = 0.008 r = 0.40; P = 0.005

r = 0.42; P = 0.02

(a) (b)

(c)

ΔMCH

ΔMCH 95% confidenceinterval


Positive correlation between DMCH and Dfat mass (kg) (a), DMCH and Dfat mass (%) (b), and DMCH and DAgRP (c) in the obese adolescents.AgRP, agouti-related protein; MCH, melanin-concentrating hormone.

Table 3 Simple linear regression analysis of Dadiponectin,Dalanine transaminase, and Dmelanin-concentrating hormoneas dependent variables in obese adolescents adjusted by ageand sex

Variables R2 P

DAdiponectinDGlycemia 0.16 0.04DInsulin (mU/ml) 0.23 0.002DHOMA-IR 0.25 0.001

DALTDAST 0.73 0.000DGGT 0.60 0.000

DMCHDFat mass (kg) 0.18 0.03DFat mass (%) 0.20 0.01DAgRP 0.22 0.007

ALT, alanine transaminase; AgRP, agouti-related protein; AST, aspartatetransaminase; GGT, g-glutamyl transpeptidase; HOMA-IR, homeostasis modelassessment-insulin resistance; MCH, melanin-concentrating hormone.



the glucose carrier by different mechanisms. It is relevant

to note that the present study showed that the combined

exercise promoted a significant change in the adiponectin

concentrations, and reinforced the anti-inflammatory

response due to this kind of exercise, resulting in greater

insulin sensitivity [38].

The above-mentioned mechanism could be observed by

the positive correlation between Dadiponectin and changes

in glycemia, insulin, and HOMA-IR (Fig. 1a–c) and

confirmed by the simple analysis linear regression, which

revealed that changes in glycemia, insulin, and HOMA-IR

were independent predictors of significant increase in

adiponectin concentration (Table 3). Similar to our data,

plasma adiponectin levels are known to be associated with

insulin sensitivity. The lower levels of plasma adiponectin

are known to be a risk factor for insulin resistance, and an

inverse correlation between adiponectin and HOMA-IR

and fasting insulin levels was observed [38].

Differences were verified in adiponectin, and leptin

levels between AT and AT + RT therapy groups at

baseline conditions. We hypothesized that this difference

occurred when the AT group presented minor values of

BMI and fat mass (kg) at the beginning of therapy.

Indeed, after 1 year of intervention, it was observed that

the AT group presented lower values of adiponectin and

leptin levels compared with the AT + RT group. This

difference could be explained by the effect of AT + RT

protocol on body composition and insulin sensitivity. As

mentioned previously, the AT alone is not sufficient to

maintain or improve lean mass during weight-loss therapy,

which can promote a decrease in metabolism and can lead

to more difficulties in obtaining success in obesity

control. AT + RT can promote maintenance of lean mass,

which can be associated with improvement in leptin

concentration and insulin action, strongly related to

adiponectin concentration, thus increasing the glucose

uptake. The improvement of adiponectin and the decrease

in leptin through treatment may be the target for prevention

of NASH. However, in our study, the AT protocol was not

enough to improve it, while the AT + RT proved to be more

effective in achieving this important clinical target in

NAFLD treatment.

Recent studies have demonstrated the efficacy of RT

programs ranging from 6 to 12 weeks in duration for

improving body composition in overweight and obese

children [39,40].

In addition, when we analyzed data by simple linear

regression, DAST and DGGT were independent predic-

tors of significant changes in ALT, while Dfat mass (kg

and %) and DAgRP were independent predictors of

significance in DMCH (Table 3).

Several studies observed a positive association between

the elevated hepatic transaminases with liver fat accu-

mulation and insulin resistance [41,42], similar to our

results (Fig. 2a–c; Table 3). Serum liver enzyme

abnormalities are primarily restricted to elevations of

ALT. Since the markers of liver damage, GGT, ALT, and

AST/ALT were correlated to the severity of the hepatic

damage, it was suggested that aminotransferase abnorm-

alities probably occur at an earlier stage, different from

GGT, which would require greater hepatic damage to be

altered [42].

Obesity was noted after chronic MCH infusion in mice,

and overexpression of MCH in homozygote mice results

in increased body weight [43–46]. These studies high-

light the importance of the genetic background of the

animals for their susceptibility to obesity. However, it is

necessary to explore and clarify the mechanisms of MCH

in humans, especially in NAFLD obese adolescents, to

improve clinical practices.

Moreover, in various physiological and experimental condi-

tions, AgRP was reported to be modulated in the same way

as NPY. For example, the addition of MCH to the

incubation medium led to the release of AgRP from

hypothalamic explants [16]. This result is in agreement

with our data, showing the influence of changes in AgRP in

the MCH concentration (Table 3). It is very unlikely that

MCH acts alone from its release sites, as it is expressed

with other neuropeptides and neurotransmitters [16].

It is relevant to explain that the food intake at baseline

did not present significant differences (data not shown).

Our results suggested that the AT + RT protocol can

decrease MCH, suggesting that this kind of exercise can

also affect and exert a role in food intake and weight.

Indeed, it can be seen in Table 2 that basal MCH is

higher already in AT than basal AT + RT, and in

each group there is a slight increase (no significance) in

MCH. These small increased MCH concentrations after

1 year can be partially explained by a decrease in leptin,

which exerts an important role in suppressing MCH

expression.

The small size of participants can be considered as a

limitation of our study. Nevertheless, we showed the

positive effects of AT + RT protocol in a long-term

interdisciplinary therapy to improve inflammatory bio-

markers of NAFLD and to reduce orexigenic neuropep-

tide concentrations in NAFLD obese adolescents for the

first time. It is important to consider, in future studies,

the generation of groups based on BMIs, which can

explain the basal differences of some metabolic para-

meters, such as leptin, HOMA-IR, and insulin levels

between obese adolescents with NAFLD and without

NAFLD, revealing the importance of combination

therapy in reducing NAFLD comorbidities.

A greater understanding of these complex mechanisms

linking obesity, exercise training, adipokines, and neuro-

peptides to NAFLD has led to new therapeutic

approaches to and preventive strategies for NAFLD



development. In conclusion, the long-term interdisci-

plinary therapy with AT + RT protocol was more

effective in significantly improving noninvasive biomar-

kers of NAFLD that are associated with the highest risk

of disease progression in the pediatric population.

AcknowledgementsThe authors thank the patients who participated in the

study and the following sources of support: AFIP, FAPESP

2006/00684-3, FAPESP 2008/53069-0, FAPESP (CEPID/Sleep

#9814303-3 S.T) CNPq, CAPES, CENESP, FADA, and

UNIFESP-EPM, which supported the CEPE-GEO Interdisci-

plinary Obesity Intervention Program.

Conflicts of interest

There are no conflicts of interest.

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