Participants and Study Design
Twenty volunteer females were selected by convenience sample, recruited among patients from Ribeirao Preto University Hospital, by disclosure in folders and social media. Inclusion criteria were as follows: age ranged from 20 to 40 years old, BMI between 30 to 40 kg/m2, sedentary lifestyle for at least 6 months prior to the study, and regular menses. Participants that reported a history of diabetes, hypertension, dyslipidemia, cancer, smoke, and any obesity-specific treatment (drugs or bariatric surgery) were excluded. Also, to avoid the possible biases due to hormonal influences, men were not included.
The prospective and controlled study lasted for 12 weeks, as shown in Fig. 1. Evaluations after 8-h fasting (anthropometric data, body composition, and blood collection) were done on the first week; adaptation to exercise and physical tests were done on the second and third weeks; intervention was done on the fourth to the 11th weeks; and in the 12th week, the first 2 days were for physical tests and after were for the blood collection (8-h fasting).
The intervention was at the University of São Paulo´s gym (Ribeirao Preto, SP, Brazil), 100% supervised, and with at least 50% attendance. The subjects, who completed the intervention, had an average 80% presence. The combined exercise training (alternating strength and aerobic exercise) consisted of 15 resistance exercises (for all the main muscle groups: chest, dorsum, biceps, triceps, deltoid, shoulders, quadriceps, gluteus, biceps femoral, rectus femoral, gastrocnemius, abdominal) for 30 s (it recommended that participants perform at least ten repetitions per exercise) alternated with 30 s of jogging [13]. The intervention had a duration of 8 weeks, with a frequency of three times/week with 55 min/day of duration and intensity of 75 to 90% of heart rate maximum (HR) and multiple repetitions maximum-RM (2 weeks of 75%, 4 weeks of 80%, and 2 weeks of 90% of HR and RM). The intensity of training was controlled by the heart rate monitor (Polar®) and the rating of perceived exertion (RPE) [14]. The same trained professional supervised all exercise sessions and the heart rate of participants. During the intervention, we emphasized to all participants to keep constant food intake. There were no diet intake restrictions.
The Research Ethics Committee of the Clinical Hospital from the Ribeirao Preto Medical School, University of São Paulo, approved the study (protocol # 1387.040/2016), and the study is in accordance with the standards of ethics outlined in the Declaration of Helsinki. All subjects gave free written consent.
Phenotypic Measures
All subjects have fasted for 8 h for evaluation. Bodyweight was measured by an electronic platform Fiziola™ scale with a precision of 0.1 kg and a maximum capacity of 300 kg. A rigid vertical shaft with 0.5 cm graduation was used to measure body height. The waist circumference was measured with an inextensible tape at the largest circumference between the last rib and the iliac crest. The body composition was evaluated with the deuterium oxide dilution method [15], each volunteer having received a dose of 1 ml/kg, 7% deuterium oxide (Cambridge Isotope®, USA). Urine samples were collected before and 3 h after dose intake. Samples were stored at − 80 °C until analysis. Deuterium enrichment in urine samples was determined by mass spectrometry (Europa Scientific Hydra System™, Cheshire, United Kingdom).
Physical performance tests were performed before and after the intervention. The aerobic performance was evaluated by an adapted, incremental shuttle walking test [16]. It required the participants to walk/run up and down a 10-m course, which started at 4 km/h, increasing 0.28 m/s every 3 min by stage. The speed at which the participant walked/ran was dictated by an audio signal and was interrupted when subjects could not maintain the determined rhythm (Additional file 1: Table S1). The estimated maximum oxygen consumption (VO2max) was determined, according to Heyward [17].
The strength performance was evaluated by multiple repetitions (RM) in the bench press (upper limbs) and squat (lower limbs) exercise for determination of maximum force (kg). After warm-up, with determined load, each participant should perform the correct movement to exhaustion or to complete ten repetitions (if exhaustion was not achieved in ten repetitions, there was a rest of approximately 10 min for the next attempt). After the test, to calculate the maximum load or RM, we used the formula by Brzycki [18].
Telomere Length Measures
After 8 h of fasting, a trained professional collected the patients’ peripheral blood in EDTA tubes. Genomic DNA was automatically extracted from the leukocyte using the Maxwell MDx (Promega Corporation®, Madison, WI) instrument and the Maxwell® Blood DNA Purification kit.
The relative quantification of TL was determined by telomere to single-copy gene ratio (T/S) from calculation: ΔCt (Ct(telomeres)/Ct(single-gene)). For the calculation of 2−ΔΔCt in this assay, each sample was normalized to the average T/S ratio of a reference sample, using the standard curve and validation sample as the reference [19]. It measured by quantitative polymerase chain reaction (qPCR), according to [20]. The thermal cycler used was the 7500 Fast Real-Time PCR System (Applied Biosystems®).
For the reaction, the SYBR Green PCR Mastermix kit (Qiagen®) was used in a final volume of 20 μL. Concentrations of telomere repeat copy number (T) and 36B4 (Ribosomal Protein Large PO) (S) as reference for the single-copy gene were 700 nM of each primer. DNA concentrations were and 20 ng (these values were selected from the standard curve). Telomere primer sequences (5′ to > 3′) were tel 1, GGTTTTTGAGGGTGAGGGTGAGGGTGAGGGTGAGGGT and tel 2, TCCCGACTATCCCTATCCCTATCCCTATCCCTATCCCTA. Beta globin primer sequences were 36B4u, CCCATTCTATCATCAACGGGTACAA, and 36B4d, CAGCAAGTGGGAAGGTGTAATCC. For quality control, a minimum of three assays was performed, and the average was used for analysis [21, 22].
Statistical Analysis
Descriptive statistics consisted of mean and standard deviation values. Data normality was verified by the Shapiro-Wilk test. Then, the appropriately paired t test or Wilcoxon test was used to compare data before and after interventions. To verify correlations, the Pearson or Spearman test was used. Associations were verified by linear regression. Statistical significance was considered at p < 0.05. All analysis was performed with SPSS™ version 20.0 software (SPSS Inc.).