The primary outcome measure is hepatic fat content, which will be assessed with the controlled attenuation parameter. The secondary outcomes of this study will test the additional effects of exercise training on traditional cardiometabolic risk factors [body composition, blood pressure, glycohemoglobin (HbA1c), serum lipids, and insulin resistance] and non-traditional cardiometabolic risk factors (visceral adiposity, endothelial function, cytokines, adipokines, aminotransferases, inflammatory biomarkers, uric acid, ferritin, albumin, and cardiorespiratory and muscular fitness). Also, we include neuronal function biomarkers as measured by neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), and brain-derived neurotrophic factor (BDNF) levels (second secondary objective). Finally, attention capacity and psychological measurements will be assessed to evaluate the additional effect of the exercise intervention on the executive function, quality of life, self-esteem, and well-being of overweight and obese participants. Other covariables of interest include pubertal development, physical activity levels, sedentary behavior, dietary assessment, demographic characteristics, and side effects (Table
2).
Table 2
Overview of the measurements and methodology at baseline and post-test in the HEPAFIT study
Primary outcome |
Hepatic fat (%) | Vibration-controlled transient elastograph |
Secondary outcomes |
Body mass (kg) | Scale |
Height (cm) | Stadiometer |
Waist circumference (cm) | Non-elastic tape |
Blood pressure | Oscillometric monitor device |
Heart rate | Oscillometric monitor device |
Lean mass (kg), total body fat (%), bone mineral density (g/cm2), abdominal adiposity (g), truncal adiposity (g), limb adiposity (g) | Dual X-ray absorptiometry |
Visceral adiposity (cm2) | Bioelectrical impedance |
High-density lipoprotein cholesterol (mg/dL), low-density lipoprotein cholesterol (mg/dL), total cholesterol (mg/dL), total triglycerides (mg/dL), serum glucose (mg/dL) | Enzymatic spectrophotometry |
Serum insulin (mU/L) | Electrochemiluminescence immunoassay |
Glycohemoglobin (mg/dL) | Electrochemiluminescence immunoassay |
Alanine (U/L), aspartate (U/L), gamma-glutamyl transferase (U/L) | Enzymatic assay |
Uric acid, albumin (mg/dL), ferritin (mg/dL) | Enzymatic assay |
hs-C reactive protein, ENA-78, GCSF, GM-CSF, GRO, GRO-α, I-309, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 p40/p70, IL-13, IL-15, IFN-γ, MCP-1, MCP-2, MCP-3, MCSF, MDC, MIG, MIP-1β, MIP-1δ, RANTES, SCF, SDF-1, TARC, TGF-β1, TNF-α, TNF-β, EGF, IGF-I, angiogenin, oncostatin M, thrombopoietin, VEGF-A, PDGF-BB, leptin, BLC, Ckß8–1, eotaxin, eotaxin-2, eotaxin-3, FGF-4, FGF-6, FGF-7, FGF-9, Flt-3 ligand, fractalkine, GCP-2, GDNF, HGF, IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IL-16, IP-10, LIF, LIGHT, MCP-4, MIF, MIP-3α, NAP-2, osteopontin, osteoprotegerin, PARC, PLGF, TGF-β2, TGF-β3, TIMP-1, TIMP-2 (mean spot pixel densities) | Proteome Profiler human cytokine array panel A kit |
NT-3, NT-4, BDNF (pg/mL) | Surface plasmon resonance biosensors |
Flow-mediated dilation | High-resolution ultrasound device |
Carotid intima-media thickness | High-resolution ultrasound device |
Cardiorespiratory fitness | 20-m shuttle run test Cardiopulmonary exercise test |
Muscular strength | Handgrip strength Standing long jump tests |
Speed and agility | 4 × 10 shuttle run test |
Flexibility | Sit-and-reach test |
Attention capacity | d2 attention test Stroop task test |
Physical activity enjoyment | Physical activity enjoyment scale |
Potential confounders |
Physical activity and sedentary behavior | Actigraph GT1M, dual-axis accelerometer ActivPal |
Dietary assessment | 24-h diet record Food frequency questionnaires |
Pubertal development (Tanner stage) | Questionnaire |
Family medical history, demographic characteristics, socioeconomic status | Questionnaire |
Secondary outcome measures
Blood samples will be obtained from each subject early in the morning by venipuncture from the antecubital vein, following a 10-h overnight fast. Blood samples will be drawn into a tube containing ~1.8 mg EDTA-K3 per mL blood for plasma and a tube with a polymer gel for serum determinations (Vacutainer, Becton Dickinson & Company 2017). Samples will be handled according to Clinical Laboratory Improvement Amendments, which must be followed to achieve valid test results that can be used for diagnoses [
37]. Blood samples will be stored at room temperature until centrifugation. Samples should undergo centrifugation immediately. This will be carried out at 1500
g for 15 min at 4 °C and will give three layers (from top to bottom): plasma, leukocytes (buffy coat), and erythrocytes. The supernatant (plasma) and serum will be carefully aspirated at room temperature and aliquoted into cryovials. Finally, both samples will be stored at −80 °C before the analysis of serum lipids, adipokines, aminotransferases, inflammatory biomarkers, uric acid, ferritin, albumin insulin, and neuronal function biomarkers using surface plasmon resonance biosensors.
Serum lipids, including high-density lipoprotein cholesterol (HDL-C), LDL-C, total cholesterol, and total triglyceride, will be measured with an automatic biochemical analyzer (Deyi Biomedical Technology Co., Ltd., Beijing, China). Concentrations of serum insulin and HbA1c will be analyzed with commercially available electrochemiluminescence immunoassay kits (Roche Diagnostics GmbH, Mannheim, Germany). The homeostatic model assessment of insulin resistance (HOMA-IR) and homeostatic model assessment of pancreatic β-cell function (HOMA-β) indices will be calculated with the following equation:
$$ \mathrm{HOMA}-\mathrm{IR}=\mathrm{fasting}\ \mathrm{insulin}\ \left(\mathrm{FINS};\mathrm{Ins}0\right)\times \mathrm{fasting}\ \mathrm{blood}\ \mathrm{glucose}/22.5 $$
and
$$ \mathrm{HOMA}-\beta =20\times \mathrm{Ins}0/\left(\mathrm{fasting}\ \mathrm{blood}\ \mathrm{glucose}-3.5\right) $$
Alanine aminotransferase, aspartate aminotransferase, and g-glutamyl transferase levels will be determined by enzymatic assays (Beckman Coulter, Brea, CA, United States). Uric acid, albumin, and ferritin will be measured in an enzymatic spectrophotometer (Beckman Coulter, Brea, CA, United States). All assays will be measured in duplicate according to the manufacturers’ standard procedures.
Non-traditional biomarkers will be evaluated for the presence and relative amounts of 80 different inflammatory markers using a Proteome Profiler Human Cytokine Array Panel A kit (R&D Systems, Minneapolis, MN) (Additional file
1: Table S1). These include: hs-C reactive protein, ENA-78, GCSF, GM-CSF, GRO, GRO-α, I-309, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 p40/p70, IL-13, IL-15, IFN-γ, MCP-1, MCP-2, MCP-3, MCSF, MDC, MIG, MIP-1β, MIP-1δ, RANTES, SCF, SDF-1, TARC, TGF-β1, TNF-α, TNF-β, EGF, IGF-I, angiogenin, oncostatin M, thrombopoietin, VEGF-A, PDGF-BB, leptin, BDNF, BLC, Ckß8–1, eotaxin, eotaxin-2, eotaxin-3, FGF-4, FGF-6, FGF-7, FGF-9, Flt-3 ligand, fractalkine, GCP-2, GDNF, HGF, IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IL-16, IP-10, LIF, LIGHT, MCP-4, MIF, MIP-3α, NAP-2, NT-3, NT-4, osteopontin, osteoprotegerin, PARC, PLGF, TGF-β2, TGF-β3, TIMP-1, and TIMP-2.
This assay will be performed according to the instructions provided by the manufacturer. Briefly, 50–200 μL of serum samples will be thoroughly suspended in 1.5 mL of array buffer 5, incubated at room temperature for 15 min, and centrifuged for 5 min at 5000g. The supernatant (1 mL each) will be added to a cocktail of biotinylated antibodies and incubated at room temperature for 1 h. The sample/antibody mixture will be subsequently incubated at 4 °C for 19 h with a membrane embedded with antibodies specific to each of the 80 different inflammatory markers analyzed. The pixel densities of each blot (band), representing the amount of each inflammatory marker present, will be determined using the ImageJ software (National Institutes of Health, Bethesda, MD). These non-traditional biomarkers will be measured in a random sub-sample (n = 10, for each group).
Surface plasmon resonance allows real-time monitoring of NT-3, NT-4, and BDNF (R&D Systems, Minnesota, USA). In a typical experiment, the signal reflection, measured at a fixed angular position, is evaluated as a function of time. The real-time quantification will use a specific anti-protein antibody/protein. All experiments will be carried out at 25 °C using a Biacore 2000 instrument with a CM5 chip (Biacore, Uppsala, Sweden). HBS-EB buffer (10 mM 4-(2 hydroxyethyl) piperazine-1-ethanesulfonic acid [HEPES], 150 mM NaCl, 3 mM ethylene diamine tetraacetic acid [EDTA], 0.005% Tween 20, pH 7.4) will be used as a continuous running buffer at a 5–60 μg/mL flow rate. The antibodies (ligand) anti-NT-3, anti-NT-4, and anti-BDNF will be immobilized onto the CM5 sensor surface at a concentration range of 10–50 μg/mL. Before immobilization of the antibodies, the sensor surface will be activated via an amino-coupling chemistry kit [1-ethyl-3-(3′-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide (GE Healthcare, Uppsala, Sweden)]. For the immobilization steps, the basic principles of assays using surface plasmon resonance will be applied, such as preconcentration and a binding assay with the analyte. Each antibody solution will be injected individually to couple with the activated surface until the appropriate immobilization level is achieved. Activated carboxyl group excess will be blocked by 1 M ethanolamine hydrochloride (pH 8.5). A reference or control flow cell with 50 μg/mL bovine serum albumin (Sigma-Aldrich, Saint Louis, MO) at pH 5.0, but no immobilizing solution, will be used to subtract the instrument systematic noise and drift (background response). After each injection of a binding assay with the analyte and standard samples, an optimal regeneration solution will be injected in agreement with previous studies [
38,
39]. The percentage of surface regeneration will be estimated using [1 − (Rreg/Ro) × 100]. If the percentage regeneration achieved is below 10%, other regeneration solutions should be injected until the percentage regeneration is higher than 50%. Standard curves will be constructed by dilution of each standard recombinant protein NT-3, NT-4, and BDNF. A range of 20–5000 ng/mL will be used to obtain the curve. Each sample of protein will be injected in triplicate.
Diluted plasma samples will be quantified and the binding response will be calculated by subtracting the response measured in the control flow cell from the response in the sample flow cell. This value is applied to the simple 1 : 1 L binding model (A + B or AB). The Langmuir model is the most commonly used model for calculating binding affinity. The report points will be taken 10 s before and 100 s after taking a measurement and the difference between these two report points will be taken as the end-point measurement.