Subjects
Healthy, trained runners (18–50 yrs.) were recruited by advertisements at local educational institutions and running clubs. Inclusion criteria were 1) maximal oxygen uptake (VO2max) > 50 ml O2 kg− 1 min− 1 for women and > 55 ml O2 kg− 1 min− 1 for men, 2) running at least 3 times a week, 3) running being the primarily training activity. Exclusion criteria were use of medicine, diagnosed metabolic diseases, injuries which hindered running and body mass index (BMI) > 25 kg m− 2. Informed written and oral consent was obtained according to the Helsinki Declaration, and the study protocol was approved by the local ethics committees in Region Midtjylland, Denmark (journal no. 1-10-72-287-13) and registered at ClinicalTrials.gov (NCT03561337).
Thirty-two subjects met the criteria for participation and were enrolled into the study randomized into two groups matched in pairs. The subjects were matched two and two for age, training history, running performance (6K TT) and maximal oxygen uptake (VO2max).
Two subjects were injured after randomization, but before the initiation of the training period. Further four subjects were injured during the intervention period. This left two subjects without a proper match, resulting in 12 matched pairs who completed the trial (22 men and 2 women).
Design
The present study was completed as a double-blinded block-randomized controlled intervention trial. All subjects followed a six-week endurance training program. During the intervention period half of the runners were randomized to ingest a PRO beverage before and PRO-CHO beverage after each exercise session (PRO-CHO). The other half of the group (CHO) ingested an energy matched CHO beverage before and after each exercise session. Prior to and following the six-week intervention period the runners performed a VO2max-test, a 6K TT, had body composition determined by bioimpedance and a resting biopsy obtained from m. vastus lateralis. Furthermore, dietary records were obtained during the first and last week of the intervention period. A schematic overview of the study design is illustrated in Fig. 1.
Intervention beverages
Intervention beverages consisted of either CHO or PRO and PRO-CHO, and were ingested within 10 min before and 10 min after each exercise session. Subjects were instructed to not consume any food or any other beverages 2 h before and after each exercise session. PRO-CHO ingested 0.3 g PRO kg− 1 (Whey PRO hydrolysate Lacprodan® HYDRO.365, Arla Food Ingredients Group P/S, degree of hydrolysis between 23–29%, Leucine content: 9.3 g pr. 100 g) before each exercise session, whereas CHO received a similar amount of energy (Maxim Energy Drink, Maxim International, Ishøj, Denmark). After each exercise session, PRO-CHO had 0.3 g PRO kg− 1 in combination with 1 g CHO kg− 1, whereas CHO received a similar amount of energy (1.3 g CHO kg− 1). The amount of CHO in the post-exercise beverages corresponded to the recommendations for CHO intake to optimize glycogen resynthesis after strenuous endurance exercise (1-1.2 g CHO kg− 1) from the American College of Sports Medicine (19). Beverages were energy matched to minimize the risk that any potential ergogenic effect of the addition of PRO to the sports beverages compared to CHO was related to an insufficient CHO intake in either of the groups or due to an extra amount of energy. The PRO-CHO beverages were added a non-caloric sweetener (Sport Citrus-Apple or Blackcurrant Fun Light, O. Kavli AS, Denmark) to mask the flavor of the added ingredients. Beverage content were packed in small powder bags by Arla Food Ingredients Group P/S and handed to the participants by lab technicians, who did not know the coding for the content. The subjects had to dilute the content of the package in water before and after each training session. Subjects were informed that the study would test different sports beverages with different compositions during the intervention period, but did not know the specific contents of the pre- or post-exercise beverages, or their potential effects on performance and recovery.
Training
The endurance training consisted of a running program individually customized to the training status of the matched pairs with the purpose of improving performance. Moreover, the training was standardized for each matched pair and therefore identical between the two groups in regard to intensity, frequency and total training volume. The training programs were designed based on training status and injury history. The programs consisted of approximately 5–7 workouts per week, of different duration and intensity.
Subjects registered their training intensity (relative workload in regard to heart rate (HR); intensities were zoned: Low = 65–80% HRmax; Moderate = 81–88% HRmax and High = 89–100% HRmax) and exercise duration by using the software program Training Peaks (www.trainingpeaks.com) and using the ‘time-in-zone’ approach (20). HR was registered by a HR monitor (RS800 or RS800CX, Polar Electro Denmark ApS), and runners used GPS during all the exercise sessions. Lastly, subjects, if needed, got permission from the research team to perform the exercise session at a cross-trainer or bike to reduce the risk of overload injuries. However, the substituted training session should be performed with the same intensity and duration as planned. No other training for the legs was allowed during the intervention period.
Determination of maximal aerobic power (VO2max) and heart rate (HRmax)
Maximal aerobic power (VO2max) was measured before the intervention as part of the screening and after the six-week intervention period. After a 10-min warm-up subjects would run for two mins at a self-detected velocity they predicted would cause exhaustion after 4–7 min with increasing slope of the treadmill (Woodway Pro XL, Woodway USA Inc., Waukesha, Wisconsin, USA). After the two mins, the slope was raised 2% every 90 sec until voluntary exhaustion. Respiratory variables were measured continuously through a mouthpiece connected to an automated metabolic cart using a mixing chamber system (AMIS 2001, Innovision, Odense, Denmark). Before each test, the gas analyzer was calibrated by a known gas solution; a high-precision two-component gas mixture of 16.0% O2 and 4.0% CO2. In addition, calibration of the flow meter was performed at low, medium and high flow rates with a 5L air syringe. Expired O2 and CO2, and the inspired minute ventilation (VE), were monitored continuously, and VO2 values were calculated and averaged during 30 sec intervals. VO2max was defined as the highest mean VO2 value obtained during a 30 sec period. To ensure that a true VO2max was attained, at least two of the following three criteria had to be fulfilled: 1) VO2max plateau was reached, 2), HR was within ± 5 beats min − 1 of estimated maximal HR (HRmax) (220-age), and 3) VCO2 (L min− 1)/VO2 (L min− 1) > 1.1.
HR was measured continuously during the test by a wireless HR monitor (RS800 or RS800CX, Polar Electro Denmark ApS) and HRmax was determined. HRmax was later used to customize the six-week training program for each individually matched pair.
6K TT performance
A 6K TT was performed on a treadmill before (Baseline; 0wk), during (Midway; 3wk) and after the intervention (Post; 6wk) to examine changes in performance. The 6K TT was performed two days after last training session or VO2max test. After a warm up (∼10 min), subjects had a small break before completing the 6K TT on a treadmill (Woodway Pro XL, Woodway USA Inc., Waukesha, Wisconsin, US) in a zero-grade position. Participants were advised to run as fast as possible. All 6K TT began with two min at a set velocity before the subject was allowed to change running speed. The start-up speed was individually determined based on performance history and standardized between tests. No music was allowed during the test and the subjects were not able to see the time during the run, but the distance. The instructor kept reminding the participant to run as fast as possible during all 6K TT, but during the last 2 km the instructor intensified the motivational support. No pre intervention beverage was ingested before the 6K TT performance test, but the post beverage was taken at the midway and final test, as part of the training program.
Nutritional status, weight and body composition
Each participant kept food diary 24 h before each test to ensure the intake was identical before each VO2max test, 6K TT and muscle biopsy, respectively. Subjects were advised to drink water, and stay rehydrated before each test. Additionally, subjects kept a food diary of their energy and macronutrient intake for four days at the beginning (wk 1) and in the end (wk 6) of the intervention period to make sure the energy and macronutrient intake did not change significantly. Logs were analyzed by MADLOG VITA ApS for total caloric intake, as well as fat, carbohydrate and protein intake excluding and including the beverages.
Subjects were instructed to ingest an energy balanced diet and stay weight stable during the intervention period. If weight changes were noted at the midway test guidance was provided to adjust the weight to the start weight at baseline.
Energy intake (EI) was validated by using Goldberg’s minimum cut-off limits for EI/basal metabolic rate (BMR) (21). BMR was calculated based on body weight, gender and age (22). The cut-off limit based on four days food registration is 1.06 (EI/BMR) for the individual reports. Reports below this value were not recognized as representative of energy balanced habitual intake and were thus excluded from further analysis (n = 1 for registration week 1 with a cut-off at 0.71, and n = 5 for registration week 6, with cut-off values ranging 0.78–1.04) (21).
Subjects were weighed at a body composition analyzer (Tanita SC330, Body Composition Analyzer, Tanita Corporation of America, Inc., Arlington Heights, Illinois, USA) in the morning before the resting biopsy was obtained.
Resting biopsy
A muscle biopsy was obtained at rest two days prior to the intervention period began and again after the intervention (two days after the last VO2max-test). All biopsies were obtained at the same time of the day in the morning after overnight fasting. Subjects were instructed to refrain from physical activity for 48 h before the biopsy. After local anesthesia (lidocaine), an incision was made through the skin and fascia, and the muscle biopsy was taken from the middle third of the lateral vastus muscle using a modified Bergström needle with suction. Biopsies were frozen directly in liquid nitrogen (N2) and stored at -80 °C until later analyses.
Western blotting analysis
The skeletal muscle biopsies were freeze-dried and proteins were purified by homogenization in homogenization buffer [20 mM Tris, 50 mM NaCl, 50 mM NaF, 5 mM tetrasodium pyrophosphate, 270 mM sucrose, 1% (vol/vol) Triton X-100, 2 mM DTT, and Proteinase inhibitor cocktail (Complete, EDTA-free; Roche Diagnostics, Indianapolis, IN)] on a Precellys 24 (Bertin Technologies, Montigny-le-Bretonneux, France). The samples were gently swirled at 4 °C for 15 min, before being centrifuged at 13,000 rpm at 4 °C for 20 min. The supernatant was collected, frozen in liquid nitrogen, and stored at -80 °C until further analysis. Protein concentrations were determined by the Bradford assay.
In short, western blotting was performed as follows; 15 µg protein was loaded onto a 4–15% SDS gel (Criterion TGX stain-free gels, Bio-Rad, Hercules, CA, USA), followed by electro blotting onto a PVDF membrane. These stain-free gels allow for the detection of total protein content; a trihalo compound reacts with tryptophan residues in an ultraviolet-induced reaction and produces fluorescence. Therefore, a picture of the membrane was taken for total protein assessment. Membranes were blocked with 2.5% skimmed milk for 2 h before the primary antibody was added and incubated overnight at 4 °C. The following primary antibodies were used: Cytochrome C (no. 4270), VDAC (no. 4661), HSP60 (no. 12165), COX-IV (no. 4850), PHB1 (no. 2426), SDHA (no. 11998) all from Cell signaling Technology, Danvers MA. Following several washes, the membrane was incubated with the secondary antibody (goat anti-rabbit IgG, no. 7074; horse anti-mouse IgG, no. 7076, Cell signaling Technology, Danvers, MA) for 1 h at room temperature. Proteins were visualized by a chemiluminescence detection system (Super signal dura extended duration substrate; Pierce).
Citrate synthase and β-hydroxyacyl-CoA dehydrogenase activity (Spectrophotometry)
Maximal activity of the enzymes citrate synthase (CS) and β-hydroxyacyl-CoA dehydrogenase (HAD) were determined in the muscle samples. Biopsies were lyophilized and freeze dried for 48 h. After dissection and removal of visible connective tissue and blood under a microscope, the muscle specimens were weighed to 1.5–2.3 mg dry weight and stored at -80 °C until further analyses. Samples were then homogenized for 3 min in 600 µl ice-cold buffer (50 mM Na2HPO4, 1 mM Ethylenediamine tetraacetate (EDTA) and 0.05% v/v Triton X-100 at pH 7.4) before they again were frozen in liquid N2, and stored at -80 °C until further analyses. In one biopsy, the sample contained so much connective tissue that the sample portion was not sufficient to obtain the same weight (0.25 mg vs. 1.5–2.3 mg dry weight), and only 100 µl buffer was used for this sample. CS and HAD activities were analyzed spectrophotometrically (Beckman DU 650 Spectrophotometer, USA) according to Gejl et al. 2014 (23). Absorbance rates were double determined and recorded for 600 sec, and expressed as µmol g dw− 1 min− 1.
Statistics
Subject characteristics were analyzed using Student’s t-test. The effects of group (CHO vs PRO-CHO) and time and their interactions were analyzed using a two-way analysis of variance with repeated measures for time. When a significant interaction or main effect was observed, all Pairwise Multiple Comparison Procedures (Holm-Sidak method) were used to evaluate a difference from baseline to post intervention within each group.
Data were tested for normality (Shapiro-Wilk normality test) and equal variance before analysis. The following data were log-transformed before statistical analysis: CD36, SDHA, COX-IV, pGS, CS, 6K TT. Protein expression data is presented as median ± upper/lower quantile and minimum and maximum. Additional data are presented as mean ± SE if not otherwise indicated. Data were analyzed in Sigmaplot (ver. 13.0, Systat Software inc. Berkshire, UK), and graphs were designed in Graph Pad Prism (Graph Pad Prism ver. 6.02, San Diego, USA).