Study design
A controlled, randomized clinical trial using the minimization method will be conducted, with blinding of those who will evaluate the outcomes and two parallel groups for the purpose of showing superiority. Sixty patients with MS and IR will be included and will undergo medical evaluation, laboratory tests, measurement of muscle mass and fiber type composition in the thigh, and cardio-respiratory physical capacity, both before and after 12 weeks of a treadmill exercise program. The participants assigned to the intervention will receive HIIT-low volume for 22 min, and the control group will receive CAE for 36 min three times per week. The study will be implemented and reported in line with Standard Protocol Items for Randomized Trials [28] (Fig. 1 and Additional file 1).
Study site
This study will be carried out in the cardiac rehabilitation and physiotherapy section of the facilities of the outpatient clinic of the IPS-Universitaria, in Medellín (Colombia).
Recruitment of participants
Volunteers will be recruited from the academic community of the University of Antioquia, associated institutions and in nearby communities, through messages sent by email or Facebook, posters located in various faculties, and through direct invitation to those who attend health promotion and cardiovascular prevention activities at the University. The expected time for the recruitment of participants will be 18 months. If the recruitment is slower than expected we will implement educational and communication strategies within the target populations, in order to increase the number of participants. Also, we can extend the recruitment period for another 6 months if needed. The recruitment will end once the number of required volunteers is obtained. There will be no incentives paid to participate in this study.
Eligibility criteria
People aged between 40 and 60 years, with three criteria for MS [1], with IR according to the homeostatic model assessment (HOMA-IR) greater than 2.25 [29, 30] and with a sedentary lifestyle [31] will be included. People with a vegetarian diet, supplemental consumption of vitamin D3, injuries or musculoskeletal diseases that prevent exercise; people in a situation of physical, sensory and cognitive disability; people with a history of cardiopulmonary disease or acute or chronic inflammatory conditions, cancer, acquired immune deficiency syndrome or diabetes mellitus; and pregnant women will be excluded.
Interventions
The training program will be controlled by physical activity specialists and will include three sessions per week of treadmill walking/running, with a progressive increase in training loads of 5% every 2 weeks and a total duration of 12 weeks.
High-intensity and low-volume interval training
The HIIT-low volume session will include a warm-up period of 3 min at an intensity of 30% of VO2 max (expressed as a measure of the external load in the speed and inclination of the treadmill), followed by six intervals that include 1 min of high intensity with a workload of 90% of VO2 max and 2 min with a workload of 50% of VO2 max. The session will end with a cooling off period of 3 min at an intensity of 30% of VO2 max for a total duration of 22 min (Fig. 2a).
Continuous aerobic exercise
The CAE session will include a warm-up period of 3 min at 30% of VO2 max, followed by 30 min at 60% of VO2 max; it will end with a cooling off period of 3 min at 30% of VO2 max for a total duration of 36 min (Fig. 2b).
Adherence to the protocol
Adherence will be established from the quantification of a percentage, taking into account the total volume of physical activity performed in relation to the total volume of physical activity scheduled.
Concomitant interventions
All people will continue with their pharmacological treatment plan and nutritional recommendations provided by the treating physician.
Measurement of covariates and outcomes
For the collection of information, electronic formats will be designed to record the data related to each training session, the measurement of covariates and the outcomes.
Clinical history and physical examination
A complete clinical history will include socio-demographic information, habits and personal and family histories. Blood pressure and heart rate will be measured in the sitting and standing positions according to current recommendations [32]. For the quantification of physical activity, the Global Physical Activity Questionnaire (GPAQ) [33] validated in Colombia [34] and applied by our research group [23], will be used. The caloric intake will be estimated from the frequency of food consumption at the beginning and end of the study.
Anthropometry
Height will be measured with a Seca® 2013 stadiometer (Seca, Germany), and body weight will be measured with an Omron® HBF-510LA scale (Omron Healthcare, Inc., USA) with accuracy of 0.1 kg. Waist circumference will be measured with a fiberglass anthropometric tape at the intermediate point between the lower edge of the last rib and the iliac crest, in the horizontal plane. The perimeter of the thigh will be measured 1 cm below the gluteal fold, perpendicular to the longitudinal axis of the thigh. Body mass index (BMI) will be calculated with the formula weight (kg)/height (m)2.
Cardio-respiratory physical capacity
VO2 max will be measured directly with an open circuit spirometer Oxycon Delta by Jaeger® (VIASYS Healthcare GmbH, Germany) and a treadmill. An exercise physiologist will monitor the test, and the electrocardiogram and heart rate will be recorded continuously. Participants will initially warm up for 3 min, walking at a speed of 3.5 miles per hour, with an inclination angle of 1%. Then, the speed of the treadmill will increase 0.5 miles per hour every minute until exhaustion. It will be considered a maximum test when (i) a plateau in VO2 despite an increase in work or a respiratory quotient equal to or greater than 1.10 are observed; and (ii) the participants reach the expected maximum heart rate.
Lipid profile and glycosylated hemoglobin
A venous blood sample will be obtained after a fast of 8–12 h to measure the total cholesterol, high-density lipoprotein cholesterol (HDL), plasma triglycerides and glycosylated hemoglobin, using standardized procedures.
Insulin resistance
HOMA-IR will be used to estimate IR, insulin sensitivity (IS) and the percentage of functioning pancreatic β cells (% β) [35]. HOMA-IR will be calculated before and between 44 and 48 h after the last session of the exercise training program, from the blood glucose and insulinemia values obtained from a venous blood sample taken after fasting for 8–12 h [35]. The measurement of insulinemia will be performed with the sandwich immunoassay technique with direct chemiluminescence technology with an ADVIA Centaur® CP Immunoassay System (Siemens, Germany).
Quantification of serum levels of musclin and apelin
Concentrations of musclin and apelin will be measured in serum using a sandwich-type enzyme linked immunosorbent assay kit (Human Musclin ELISA kit, LifeSpan BioSciences Inc., Catalog number LS-F7799, USA; Human Total Apelin ELISA kit, MyBioSource, Catalog No. MBS-725907, USA). Samples will be taken before and between 44 and 48 h after the last exercise session. Initially, the samples will be stored at − 80 °C, and then for the assay, they will be thawed, and 100 μL will be applied in duplicate to the wells of the ELISA kit. The samples will be incubated at 37 °C; subsequently, the secondary antibody conjugated to biotin will be applied; finally, the samples will be incubated with avidin coupled to an enzyme with peroxidase action. The optical density of each well will be determined in an ELISA reader (Thermo Scientific Varioskan Lux, USA) at 450 nm. The final values will be reported in picograms/milliliter of musclin and apelin.
Measurement of muscle mass
This step will be performed at the beginning and at the end of the exercise training program, within the first 96 h after the last session, by employing the dual energy x-ray absorptiometry (DXA) technique using a Discovery Wi DXA system® (Hologic, USA) and the Hologic APEX v4.5.3 program (Hologic, USA). The measurement of thigh muscle mass will be made on the right side according to the technique described by Visser and defined by an area located between two horizontal lines located at the lowest point of the ischial tuberosity and at the knee joint as the upper and lower limits, respectively [36].
Evaluation of the fiber type composition of the right vastus lateralis
The estimation of the proportion of the area occupied by type II muscle fibers will be made at the beginning and the end of the exercise training program in the first week after the last session, based on the quantification of carnosine in the right vastus lateralis muscle (VLM) using the proton magnetic resonance spectroscopy technique (1H–MRS) [37] and a flexible coil according to the technique standardized by our group to evaluate body segments of large muscle mass [38], in turn modified from Baguet’s technique [37]. A three-Tesla Magnetom Skyra magnet, with a Flex Large 4A3T coil interface and the SyngoMR D13 program (Siemens, Germany), will be used. Each person will be placed in the supine position on the spectroscopy table, with the lower limbs extended and supported on a 5-cm cushion in the popliteal region. The largest mass of the VLM will be sought in the area between the middle of the externally facing right thigh and the middle third and distal junction of the same thigh; these anatomical points will be indicated with vitamin E capsules. A voxel of 10 × 15 × 35 mm will be positioned in the most voluminous region of the muscle, in the area delimited by the capsules, using a T1-weighted gradient-echo sequence in the three planes, with 5 mm sections and repetition time (RT)/echo time (ET) = 250/2.52 ms. The acquisition of the spectra will be performed using a point-resolved single voxel spectroscopy (PRESS) sequence with and without water saturation to evaluate the carnosine and water signals, respectively. For the processing of the signals and the absolute quantification of carnosine, the jMRUI 5.1 program (Autonomous University of Barcelona, Spain) will be used, with phase correction, Gaussian apodization, adjustment of the water reference to 4.7 ppm and subtraction of water, lipids and other metabolites within the signal.
Sample size
A total required sample size of 30 people in each group was calculated assuming an IS increase of 10% [39], 15% standard deviation [5], a 95% confidence level, 80% power, a 1:1 ratio, and a dropout rate of 5%. In addition, a correlation coefficient of 0.44 between the baseline and final IS values was taken into account [40].
Random assignment, implementation and masking
The Coordinating Center of the SICOR Clinical Trial, external to the group of researchers, will perform the randomization at a ratio of 1:1 (HIIT-low volume, n = 30; CAE, n = 30), with the minimization method [41] with Minimpy® software, version 0.3 [42], taking into account the values of the following variables: age (< 50 and ≥ 50 years), gender (male and female) and BMI (< 30 and ≥ 30 kg/m2). A base probability of 0.7 will be used under the “bias coin” method and variance as a measure of the imbalance distance between the groups, for the inclusion of the next participant [43].
The communication between the researchers who will perform the intervention and the center responsible for the assignment to the treatment groups will be conducted by telephone to guarantee independence. The measurement of outcomes will be carried out by evaluators who will not know the assignment of patients to the treatment groups. Two researchers will feed a database based on information recorded in formats at each training session, with the measurement of covariates and outcomes. The files of the participants will be stored in numerical order in a safe place and will be accessible through a password. A code will be used that will prevent identification of the patient and that, in turn, will not be visible to the person performing the statistical analysis.
Statistical analysis
Initially, an exploratory analysis of the data will be carried out, and the Shapiro-Wilk test will be used to evaluate if the quantitative variables come from a population with a normal distribution. The mean and the standard deviation, the median and the interquartile range and the percentage will be used for the description of the socio-demographic, clinical, anthropometric and physiological variables of the included persons.
Both intention-to-treat and by-protocol analyses will be carried out; the latter will consider those patients who had adherence higher than 80%. The multiple imputation method (10 imputations) will be used to manage missing data based on age, gender, BMI and baseline VO2 max [44].
Analysis of covariance (ANCOVA) will be used [45] to estimate the effect of HIIT-low volume compared to CAE on IR, IS, percentage of pancreatic β-cell functioning, plasma levels of musclin and apelin and mass and composition of thigh muscle by fiber type at the end of the intervention. The assumptions of homogeneity of variance will be assessed with the Levene F test and the linearity and homogeneity of the slopes. If the assumptions are not met, a logarithmic transformation will be performed, or the Box-Cox procedure will be used. The variables used in the minimization procedure (age, gender and BMI) will be included in the analysis according to the current recommendations [41]. A re-randomization test will be performed using permutations for inference purposes [41].
To establish if the musclin, apelin, muscle mass and fiber type composition of the thigh mediate the effect of training on IR, a theoretical model was proposed to estimate the total, direct and indirect effects and the percentage explained by the mediator (Fig. 3). A model of structural equations will be used with the “path analysis” technique and basic principles proposed in the context of clinical trials [46,47,48]. A statistical significance level œ = 0.05 will be established, and STATA, version 14.0, and IBM SPSS Statistics, version 21.0, will be used.
Data monitoring
A data monitoring committee will not be formed because the duration of the intervention is short, the risk is minimal, and no interim analysis is required.
Adverse events
The training modalities that are going to be used are safe; the reported frequencies of adverse events are very low and are mainly related to osteo-muscular injuries [6, 7]. The possible adverse osteo-muscular, metabolic, cardiovascular and other types of adverse events that occur during physical training will be monitored. The physical activity specialist in charge of the program will investigate and record the possible clinical manifestations related to the exercise.
Audit
An independent Audit Committee will be made up of two professionals: a specialist in sports medicine and an epidemiologist. This committee will verify every 6 months the processes related to recruitment, informed consent, eligibility, assignment to the treatment groups and adherence to the intervention.