Subjects
Healthy men (N = 7; means ± SD: 24.9 ± 2 yrs) without cardiovascular, metabolic or respiratory disease were recruited from the general public and completed all exercise trials. All subjects were recreationally active and participated in sports/exercise for a minimum of 30 min, at least three times a week. None of the subjects were taking nutritional supplementation for weight gain or weight loss, nor taking supplemental vitamins or minerals.
Physical activity monitoring
All subjects recorded their habitual physical activity on the short version of the international physical activity questionnaire (I-PAQ). Further, subjects were asked to refrain from any form of recreational and competitive sports or exercise training during the entirety of this study, except for the exercises prescribed in the study. Weekly expended metabolic equivalents (METS) were calculated based on formulas designed by Craig et al. (30), and included energy expenditure for walking, moderate physical activity and vigorous physical activity.
Diet monitoring
Subjects were asked to adhere to a self-selected weekly diet, which was kept the same for three weeks. Each subject recorded his daily food intake in a food diary throughout the entire study. Finally, subjects fasted overnight (allowed plain water) prior to each trial visit. The energy and nutrient composition of the diets were assessed using an interactive nutrient analysis tool designed by the Singapore Health Promotion Board (http://focos.hpb.gov.sg/eservices/ENCF/).
Baseline cardiorespiratory and strength tests
A VO2 peak test and 10-repetition maximum (10-RM) test were administered at baseline, a week prior to the first exercise training session, in order to determine cardio-respiratory fitness and muscle strength, respectively.
VO2 peak Test
Each subject warmed up on a treadmill (4Front, Woodway, Waukesha, WI, USA) by jogging at a self-selected speed (5–9 km/h), at 1% incline for 10 min. This was followed by 5 min rest before commencing the maximal aerobic test. The starting speed was 10 km/h and increased every 3 min by 1 km/h. Metabolic gas exchange during exercise was monitored continuously with a metabolic cart (Parvomedics TrueOne 2400, Sandy, UT, USA). Heart rate was monitored continuously with a heart rate monitor (Polar RS800CX, City, Finland) and ratings of perceived exertion (RPE) was determined during the last 15 s of each stage. The test was terminated upon volitional fatigue. The subjects were considered to have achieved their maximal aerobic capacity if any three of the following criteria were met:
1) Age-predicted maximal heart rate (220 – age)
2) Respiratory exchange ratio (RER) of > 1.1
3) Volitional fatigue
4) Rating of perceived exertion (RPE) > 17 (6–20 point scale)
Muscular endurance test
Each subject rested for 20 min after the VO2 peak test. Thereafter, he performed a 10-repetition maximum (10-RM) test of the following exercises in sequential order: bench press, shoulder row and squat. The bench press and squats were performed with an Olympic barbell, and the shoulder row was performed using dumbbells. When the subject was able to complete more than 10 repetitions of each exercise, he was given a 5-min break before performing another round of 10 repetitions with a heavier weight. The final 10-RM for each subject would be the heaviest weight lifted with proper form, for 10 repetitions.
Exercise training protocol
One week after baseline testing, subjects were prescribed an exercise training protocol spanning 3 consecutive weeks, with 3 consecutive days of training per week (Fig. 1). Days 1, 4, and 7 represent the first day of each week of exercise training, whereas Days 3, 6, and 9 represent the third and last day of each week’s exercise session. Subjects also arrived 24 h after the last exercise training session in week 3 (Day 10), where a final blood and saliva sample were obtained with the subjects in a seated position (24 H).
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In week 1, each subject warmed up by jogging on a treadmill for 5 min. Thereafter, he ran on the treadmill at a speed corresponding to 80% of his maximal heart rate (pre-determined during VO2 peak testing) for 5 min. This training intensity corresponded with the first ventilatory threshold (VT1) and reflects an intensity of “somewhat hard” to “hard” on the RPE scale (31). A 2 min rest was given after the treadmill run. After the rest, a series of resistance exercises (bench press, shoulder row, squats) was performed. Each resistance exercise was performed for 10 repetitions at 70% of 10-RM, with a 2 min rest between each exercise. This series of exercises (i.e. run + resistance exercise) constituted one set of the exercise protocol. Each subject completed a total of 4 sets of exercises. Water was consumed ad libitum for all sessions.
The treadmill speed and number of repetitions for the resistance exercises were increased by 10% each week. In addition, the rest interval between each exercise set was shortened from 2 min to 1 min in weeks 2 and 3. We increased the training load weekly to prevent any adaptation to the prior week’s exercise.
All exercises were performed under ambient conditions (22–25 °C) in the human performance laboratory at the Singapore Sports Institute.
Heart rate and metabolic gas monitoring
Heart rate was monitored continuously (Polar RS800CX, Polar Electro, City, Finland) from rest (seated for 5 min) until each subject completed his daily exercise session. Metabolic gases were monitored during the first and fourth set of treadmill running at each session. Heart rate (HR), VO2 and RER were measured every minute for 5 min during the 1st and 4th sets of treadmill running. We report only the 1st and 4th set of metabolic data to track changes in exercise intensity during each training session from weeks 1–3.
Blood sampling and bioassays
Blood was obtained from the antecubital vein on day 1 (first training day of week 1), day 6 (last training day of week 2), day 9 (last training day of week 3) and day 10 (24 h recovery). On days 1, 6 and 9, blood was drawn after 5 min of seated rest (Pre), immediately after exercise (Post) and after 30 min of recovery (30 min). Blood was collected into K3EDTA vacutainer tubes (Vacuette, Greiner Bio-One, Austria). Tubes were centrifuged at 2000 rpm for 10 min at 25 °C and plasma samples were extracted immediately and stored on ice, before samples were transported back to DSO National Laboratories within 6 h. On day 10 (24 h), blood was drawn from the antecubital vein after the subject had been seated quietly for at least 5 min.
All plasma samples were stored at -80 °C until subsequent analyses. EDTA blood for flow cytometry experiments were kept at room temperature for a maximum 6 h before analysis. We were unable to obtain blood from one subject at Post on day 9, hence all biomarker results were reported for six subjects.
Alarmin assays
Plasma samples were quantified using commercial ELISA kits for the following alarmins: HMGB1 (IBL, Germany), HSP70 (Cloud-Clone Corp, USA), S100A8/A9 (BioVendor, Czech Republic) and soluble receptor advanced glycation end product (sRAGE) (Abcam, U.K). Assays were performed according to manufacturers’ instructions. Experiments for each alarmin were repeated at least once, except for HSP70, which was performed once. Subject samples were randomly assayed in duplicates, with intra-assay coefficients of variation (CV) as follows: HMGB1 (9.6%, 8.6%), S100A8/A9 (9.7%, 3.5%), sRAGE (8.7%, 6.5%), HSP70 (6.3%). The assay sensitivity of each kit was: 0.2 ng/mL (HMGB1), 0.22 ng/mL (S100A8/A9), < 3 pg/mL (sRAGE), < 1.25 ng/mL (HSP70). Plasma concentrations of the alarmins were quantified with a microplate reader (Spectrostar Nano, BMG Lab Tech, City, Country).
Multiplex cytokine and chemokine assays
Cytokines (IL-10, IFN-γ) and chemokines (IL-8, MCP-1) were quantified using custom-made ProcartaPlex® kits (Affymetrix Inc, City and/or State, U.S.A). The magnetic beads were quantified using a Luminex® 200 reader (Bio Rad, U.S.A) with the MasterPlex® CT software (v1.0) with the following settings: i) sample size: 50 µL, ii) DD gate: 5,000–25,000, iii) timeout: 45 s, iv) bead event: 100. Data analysis was performed with the MasterPlex® QT software (v2.0) with logistic 4-point weighting for standard curve fitting. Endogenous concentrations of biomarkers in the sample were determined from this standard curve. The assay sensitivity of each biomarker of interest was: 2.71 pg/mL (IL-8), 2.49 pg/mL (IL-10), 11 pg/mL (IFN-γ), and 1.29 pg/mL (MCP-1).
Quantification of monocyte subsets
Absolute cell counts were assessed using a single-platform lyse-no-wash and flow count bead procedure (32). Briefly, 100 µL of EDTA whole blood were added by reverse pipetting to BD TruCOUNT™ tubes (BD Biosciences, City and/or State, USA) and incubated for 15 min at room temperature (RT) with the following monoclonal antibodies: CD14, CD16 and CD45, conjugated to anti-Allophycocyanin (APC), Phycoerythrin (PE) and Fluorescein isothiocyanate (FITC), respectively. Erythrocytes were lysed with 450 µL of 1x FACS lysing solution per tube, vortexed and incubated in the dark at RT for 15 min before acquisition with the FACS Canto II flow cytometer (Becton Dickinson, City and/or State, USA). Compensation was adjusted using three single-stain on a control sample. A control sample was obtained from a healthy male volunteer who was not part of the study, under fasted conditions. TruCount beads, CD45+, CD14+CD16- and CD14+CD16+ monocyte cell populations were gated with the FACS diva software (version 6.1.3). Acquisition was set at 10,000 counts in the CD14+16- monocytes gate (Fig. 2) and the number of events recorded was used to calculate the absolute cell number according to the equation: (number of events in region containing cell/number of events in absolute count bead region) x (number of beads per test/test volume).
-----Insert Fig. 2-----
Plasma creatine kinase activity
The enzymatic activity of creatine kinase in plasma was tested with a commercial kit (ABNOVA, City, Taiwan) and plasma concentrations were quantified with a microplate reader (Spectrostar Nano, BMG Lab Tech, City, Country) according to the manufacturer’s instructions.
Salivary cortisol assays
Saliva samples were collected by passive drooling into cryovials (Salimetrics®, Carlsbad, CA, USA) on the same days as phlebotomy: days 1, 6, 9 and 10. Samples were stored at 4 °C and transported to DSO National Laboratories within 6 h and stored in aliquots at -80 °C until further analyses. Salivary cortisol concentrations were detected with a cortisol ELISA kit (IBL, City, Germany) and quantified with a microplate reader (Spectrostar Nano, BMG Lab Tech, City, Country) according to the manufacturer’s instructions.
Statistics
Two sets of metabolic data (HR and VO2) were collected daily from the 1st and 4th set of treadmill running. A one-way repeated measures ANOVA was used to determine significant differences in mean HR and VO2 during 3 weeks of training. A 2-way repeated measures ANOVA was used to determine the effects of: i) increasing exercise intensity (weeks 1, 2 and 3) and ii) time (Pre, Post, 30 min) on plasma concentration of biomarkers. When main effects for either intensity, time or interaction were statistically significant (P < 0.05), post hoc Tukey’s multiple correction testing was used to assess mean differences within factors. A paired t-test was used to determine mean differences in biomarker concentrations between Pre (Day 1, week 1) and 24 H (Day 10, week 3). To determine the effect sizes (η2) of exercise training on alarmin response, sum of squares was divided over the total sum of squares. Effect sizes of η2 ~ 0.2, η2 ~ 0.5 and η2 ~ 0.8 were considered “small”, “medium” and “large”, respectively (33). Linear regression was used to assess the relationship between absolute or relative changes (Δ) in exercise-induced alarmins with i) MCP-1 and ii) monocyte subsets. Statistical significance was set at P < 0.05. Analyses were performed with Graphpad Prism (version 6.0.5) with data presented as means ± SD.