Experimental design and participants
Patients performed a single bout of exercise before and 3 months after Roux-en-Y Gastric Bypass. Glucose, insulin, inflammatory markers, and gut hormones were assessed before, immediately after and 30 minutes after the exercise session at each time point (Fig. 1).
Thirteen patients recruited from the Metabolic and Bariatric Surgery Unit of the Clinical Hospital of the School of Medicine of the University of Sao Paulo participated in the study. Inclusion criteria were as follows: women who were eligible for bariatric surgery (body mass index (BMI) > 40 kg/m2 or ≥ 35 kg/m2 with associated co-morbidities), 18–55 years, and not engaged in an exercise training program for at least one year before the study. Exclusion criteria involved cancer in the past 5 years, and any cardiovascular diseases, neurological disorders, or skeletal muscle impairment that would contraindicate exercise. None of the patients were receiving hormone replacement therapy.
This study is nested within a randomized controlled trial registered at clinicaltrials.gov as NCT02441361. The study was approved by the local ethical committee and all the subjects signed informed consent before participation.
Single bout of acute exercise
After a 12-hour overnight fast, patients attended our intra-hospital laboratory for the acute exercise session. Initially, an intravenous catheter (BD Saf-T-Intima™, Becton Dickinson, Utah, USA) was placed in an antecubital vein for blood sampling. Then, patients received 170 ml (209 kcal) of a mixed meal drink (Nutrison Energy®, Danone, Paris, France), with energy intake derived from protein (14%), carbohydrate (56%), and fat (30%) 30 minutes before exercise session.
At baseline, immediately after and 30 minutes after the exercise session, blood samples were collected in sodium fluoride, EDTA coated, and RST serum tubes, centrifuged at 1000g for 15 min at 4°C, and aliquoted at -80°C until analysis of glucose, insulin, inflammatory markers (i.e., Interferon gamma [INFγ], Interleukin 1 beta [IL-1β], Interleukin 1 receptor antagonist [IL-1ra], Tumor Necrosis Factor alpha [TNFα], Monocyte Chemoattractant Protein-1 [MCP1], Interleukin 6 [IL-6], Interleukin 8 [IL-8], Interleukin-17A [IL-17A], Interleukin 4 [IL-4], and Interleukin 10 [IL-10]) and gut hormones (i.e., Ghrelin, Gastric Inhibitory Polypeptide [GIP], Glucagon-Like Peptide 1 [GLP-1], Pancreatic Polypeptide [PP], Peptide YY [PYY]). For gut hormones analysis, 50 µL of protease inhibitor (Pefabloc® SC Plus, Roche) was added to blood samples collected in RST serum tubes to prevent the degradation of gut hormones, as suggested by the manufacturer. The acute exercise session comprised a 5-min warm-up followed by resistance exercises (i.e., leg press, bench press, half squat, lat pull-down, seated row, and calf raise), consisting of 3 sets of 8–12 RM with a 2-minute rest interval between sets. Then, patients performed 30-min of aerobic exercise on a treadmill at an intensity corresponding to 50% of the delta difference between ventilatory anaerobic threshold and the respiratory compensation point determined during a maximum graded exercise test 14.
Plasma glucose was assessed by a colorimetric enzymatic assay (Bioclin, Belo Horizonte, Brazil) and insulin was evaluated by a human-specific RIA method (Diagnostic Products Corp., Los Angeles, CA). Inflammatory markers and gut hormones were quantified using Luminex xMAP technology (Milliplex Map Human Bone Magnetic Bead Panel, EMD Millipore, Billerica, MA, USA). To prevent the degradation of gut hormones, a protease inhibitor (Pefabloc® SC Plus, Roche) was added in the blood samples.
Body Weight and Composition
Body weight was assessed on a calibrated digital scale and height was evaluated with the aid of a stadiometer, from which BMI was calculated. All patients underwent a whole-body dual-energy x-ray absorptiometry (DXA) scan (GE Healthcare®) to quantify fat mass and lean mass using CoreScan™ software. All DXA measurements were carried out by the same trained technician.
Aerobic capacity and muscle function
Maximum graded exercise test was performed on a treadmill (Centurion 200, Micromed), with increments in velocity or grade at every minute until volitional exhaustion. Heart rate (HR) was continuously recorded at rest, during exercise and recovery using a 12-lead ECG (ErgoPC Elite, Micromed, Brazil). Oxygen uptake (VO2) and carbon dioxide output (VCO2) were obtained through breath-by-breath sampling and expressed as a 30-second average using an indirect calorimetric system (Cortex, Metalyzer IIIB, Leipzig, Germany). The test was considered maximal when one of the following criteria was met: VO2plateau (i.e., < 150 ml/min increase between two consecutive stages); respiratory exchange ratio value above 1.10; heart rate no less than 10 beats below age-predicted maximal heart rate. Heart rate was continuously recorded at rest and during exercise and recovery. VO2peak and ventilatory thresholds (ventilatory anaerobic threshold and respiratory compensation point) were determined as previously described (14).
Lower- and upper-limb maximal strength was assessed from the leg-press (1-RM leg-press) and bench-press (1-RM bench-press) using standard equipment (Nakagym, Sao Paulo, Brazil), as previously described 15.
Statistical analyses
Data are presented as mean ± standard deviation (SD), and 95% confidence interval (CI), unless otherwise indicated. Data normality was checked through visual inspection and Shapiro-Wilk test.
Paired t-tests were utilized to compare body composition parameters (i.e., body mass, BMI, fat mass and fat-free mass), muscle function (i.e., 1-RM leg-press and 1-RM bench-press) and aerobic capacity (i.e., Ventilatory thresholds and VO2peak) before and after RYGB.
A repeated-measures one-way analysis of variance (ANOVA) was employed to examine the glucose, insulin, gut hormones (i.e., Ghrelin, GIP, GLP-1, PP, PYY), and inflammatory markers (i.e., INFγ, IL-1β, IL-1ra, TNFα, MCP1, IL-6, IL-8, IL-17A, IL-4, and IL-10) responses to an acute exercise session before and 3 months after Roux-en-Y Gastric Bypass. In case of significant F-values, post-hoc tests with Tukey’s adjustment were performed for multiple comparisons (SAS 9.2®). Second, the area under the curve (AUC) for glucose, insulin, gut hormones (i.e., Ghrelin, GIP, GLP-1, PP, PYY) and inflammatory markers (i.e., INFγ, IL-1β, IL-1ra, TNFα, MCP1, IL-6, IL-8, IL-17A, IL-4, and IL-10) were calculated using a trapezoid model, considering all time points (baseline, immediately, and 30 minutes after the exercise session). Subsequently, paired t-tests were conducted to assess potential differences between conditions (before and after surgery) for all dependent variables. Significance level was set at P < 0.05. All analyses were performed in the statistical environment R (version 3.5.3; R Core Team 2020).