Supplement with whey protein hydrolysate in contrast to carbohydrate supports mitochondrial adaptations in trained runners

Healthy, trained runners (18–50 yrs.) were recruited by advertisements at local educational institutions and running clubs. Inclusion criteria were 1) maximal oxygen uptake (VO_2max) > 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 (6 K TT) and maximal oxygen uptake (VO_2max).

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).

Intervention beverages consisted of either CHO or PRO and PRO-CHO, and were ingested within 10 mins before and 10 mins 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 and 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 [18]. 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.

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 [19]. 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 on 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.

Maximal aerobic power (VO_2max) 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 s 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 5 L air syringe. Expired O₂ and CO₂, and the inspired minute ventilation (VE), were monitored continuously, and VO₂ values were calculated and averaged during 30 s intervals. VO_2max was defined as the highest mean VO₂ value obtained during a 30 s period. To ensure that a true VO_2max was attained, at least two of the following three criteria had to be fulfilled: 1) VO_2max 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 [20].

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.

A 6 K TT was performed on a treadmill before (Baseline; 0wk), during (Midway; 3wk) and after the intervention (Post; 6wk) to examine changes in performance. The 6 K TT was performed 2 days after last training session or VO_2max test. After a warm up (∼10 min), subjects had a small break before completing the 6 K 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 6 K 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 6 K TT, but during the last 2 km the instructor intensified the motivational support. No pre intervention beverage was ingested before the 6 K TT performance test, but the post beverage was taken at the midway and final test, as part of the training program.

Each participant performed a food diary 24 h before each baseline test and repeated the recorded food intake 24 h before the midway and posttests to ensure the intake was identical before each VO_2max test, 6 K TT and muscle biopsy, respectively. Subjects were advised to drink water, and stay rehydrated before each test. Subjects were not allowed to ingest any type of dietary supplements from the time of inclusion to the study to the end of the period. Additionally, subjects kept a food diary of their energy and macronutrient intake for 4 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 4 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’ height were measured using a stadiometer and bodyweight and fat% was measured using a body composition analyzer (Tanita SC330, Body Composition Analyzer, Tanita Corporation of America, Inc., Arlington Heights, Illinois, USA) in the morning in the postabsorptive state after overnight fasting before the resting biopsy was obtained before and after the intervention.

A muscle biopsy was obtained at rest 2 days prior to the intervention period began and again after the intervention (2 days after the last VO_2max-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.

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).

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 s, and expressed as μmol g dw^{− 1} min^{− 1}.

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, 6 K 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).

No effect of the intervention was observed in Glycogen synthase (GS), cyclic ADP ribose hydrolase (CD38), and PHD (data not shown).

VO_2max was significantly increased following the training period (+ 2.4 ± 1.0%, p < 0.05) (Effect size: PRO+CHO d = 0.16, CHO d = 0.21), but was not influenced by the intervention (interaction p = 0.87) (Fig. 4b). Similarly, the runner’s fitness level (VO_2max kg^{− 1}) tended to be higher after the training period (+ 2.0 ± 1.1% p = 0.08) (Effect size: PRO+CHO d = 0.24, CHO d = 0.30), but the improvement was not influenced by the intervention (interaction p = 0.78, data not shown).

Body weight did not change during the intervention period (PRO-CHO: 0.4 ± 0.3 kg, CHO:0.2 ± 0.4 kg, time p = 0.27, interaction p = 0.79). Furthermore, body fat percentage did not change during the intervention period (PRO-CHO: 0.1 ± 0.4%, CHO: − 0.3 ± 0.3%, time p = 0.76, interaction p = 0.55).

The muscle biopsies obtained at rest after overnight fasting pre and post the intervention were analyzed for several mitochondrial target proteins. The amount of Cyt C was significantly increased in PRO-CHO compared to CHO following the intervention period. Cyt C is a protein localized within the mitochondrial intermembrane space and is important for the transport of electrons in the respiratory chain. In addition to Cyt C, several other mitochondrial proteins followed a similar non-significant pattern (SDHA, COX-IV, VDAC, HSP60, PHB1 (interaction p values between 0.07 and 0.13). These data suggest that consuming protein before and after exercise may enhance the translation of specific mitochondrial proteins to support mitochondrial biogenesis in response to exercise compared to ingestion of carbohydrate only.

Mechanistic studies investigating the impact of consuming protein with CHO compared to CHO on mitochondrial adaptations show divergent results possibly due to methodological limitations. Two studies observed no significant difference in mitochondrial MPS (post 4 h) when consuming CHO vs. CHO-PRO following prolonged endurance cycling and sprint intervals [8, 11]. However, the 4-h time course may be too short to detect the rise in mitochondrial MPS, which is supported by data from Di Donato et al. showing a rise in mitochondrial MPS 24-28 h following intense endurance exercise, but not 0.5–4.5 h post exercise [12]. Intriguingly, Churchward-Venne et al. brings new life to the debate with their recent study, investigating how different doses of dietary PRO co-ingested with CHO influence synthesis of myofibrillar and mitochondrial MPS [10]. In line with previous studies, they report no difference in mitochondrial MPS in response to none or an increasing dose of protein. However, by labeling the protein ingested with a L-[1–¹³ C]-phenylalanine, the research group demonstrated that incorporation of dietary protein-derived L-[1–¹³ C]-phenylalanine into de novo synthesis of mitochondrial protein increased dose-dependently after ingestion of 15, 30 and 45 g of PRO at 360 min post exercise [10]. In support of a positive interaction between consuming protein post exercise and mitochondrial adaptations, Hill et al. demonstrated that a 2-week dietary intervention of co-ingestion with CHO-PRO compared to CHO, resulted in significantly greater increases in PGC-1α 6 h after endurance exercise [13]. PGC-1α is a key regulator of mitochondrial biogenesis and control the translation of mitochondrial protein [24]. Therefore, these findings may suggest that PRO supplementation play a role in mitochondrial biogenesis through enhancing the exercise-induced increase in PGC-1α expression. Nevertheless, we did not detect any change in PGC-1α mRNA expression in the resting post biopsy compared to baseline (data not shown), which may be due to the biopsy being taken 48 h after the last training session.

To our knowledge, only two long-term training studies have compared biomarkers of mitochondrial biogenesis in subjects consuming PRO or placebo following exercise and prior to sleep [15, 16] (either isocaloric CHO or non-caloric placebo). Roberson et al. used a non-invasive NIRS device and observed a tendency for mitochondrial capacity to increase with training, but no difference between PRO and non-caloric placebo [16]. Knuiman et al. assessed CS and Cyt C enzyme activity and found that increases in CS activity tended to be greater in the PRO compared to CHO group [15]. However, differences in methodological approach [16], biopsy timing [15] and the comparison of PRO vs. placebo instead of CHO-PRO vs. CHO makes it difficult to compare findings. Nevertheless, current evidence is contradicting, but both mechanistic and long-term training studies have found trivial or positive effects of consuming protein on mitochondrial biogenesis [10, 13, 15]. Our data shows greater increases in specific mitochondrial proteins when consuming PRO in addition to CHO over 6 weeks of endurance training. These adaptations are likely the result of a greater accumulative increase in mRNA transcripts encoding mitochondrial proteins, which have resulted in a greater or more sustained signal when adding fast absorptive protein close to exercise and excluding pre-exercise CHO over a prolonged training period [24]. Thereby, our findings add new supporting knowledge of a beneficial effect of protein supplementation on top of previous observations demonstrating that protein feeding before and during CHO-restricted training does not impair FFA mobilization [25] or impair activation of the AMPK signaling cascade [26], but may improve net protein balance [27] and induce a more anabolic environment through mTOR activation [28]. Nevertheless, in the present study, we had no control group receiving a non-caloric beverage pre-exercise. For this reason, we are unable to tell whether the differences observed in mitochondrial proteins are a long-term response to the exclusion of CHO pre-exercise, rather than the inclusion of PRO [1, 29].

There is strong evidence to support that the ingestion of CHO prior to and during exercise improves endurance performance in the short term. However, a growing body of literature investigating carbohydrate periodization or training low, demonstrates that a single bout of exercise or short term (3–10 weeks) training programs performed with (or a portion of workouts) low muscle glycogen or low exogenous CHO availability enhance mitochondrial adaptations to a greater extent than training with normal/high CHO availability [1, 24, 29]. Intake of glucose during exercise have been shown to attenuate the rise in AMPK, a kinase recognized as the metabolic master switch in muscle glucose and fat metabolism [30], as well as to suppress lipolysis and reduce the expression of genes involved in fatty acid transport and oxidation [31]. Accordingly, carbohydrate periodization training studies investigating changes in protein expression levels have found greater Cyt C [32], and HAD [33] protein content when using a train-low model compared to control (training twice every second day vs. once daily for 3 weeks or training fasted vs. with CHO for 6 weeks). HAD is recognized as a key enzyme in the beta-oxidation of fat metabolism located within the mitochondrial matrix. It is generally well-recognized that endurance training reduces the reliance of CHO and increases fat oxidation when exercising at same absolute intensity, as training status is improved, with one key adaptation being increased HAD protein and activity [34]. In the present trial, we included already trained individuals, which may partly explain why HAD activity and CS activity (an enzyme responsible for catalyzing the first reaction in the citric acid cycle) did not increase in response to training. In fact, post hoc analysis revealed that HAD activity was decreased in the CHO group, which indicates a decreased ability to oxidize free fatty acids after the training period. It is well known that substrate utilization is strongly influenced by both short- and long-term diet changes [34]. Moreover, ingestion of CHO before and during exercise results in a marked reduction in fatty acid oxidation, and long-term consumption of a high-fat diet increase fat-oxidation and may increase HAD activity [34, 35]. Furthermore, training studies investigating the influence of CHO availability on mitochondrial enzyme activity, demonstrates that subjects who follow a ≥ 3-week train low model experience greater increases in HAD activity [32, 36–38], CS activity [32, 37], and SDHA activity [39] compared to subjects training with normal glycogen/CHO availability. In the present study, we did not manipulate or control glycogen availability. Furthermore, previous pre-exercise nutrition habits may influence this training adaptation and dependent on whether or not an individual is used to consume fast digestive carbohydrates before exercise. However, our data suggest that consuming pre-exercise CHO continuously over a prolonged training period may decrease the activity of important enzymes involved in fat oxidation and seems to reduce mitochondrial adaptations, which thus may reduce the ability to utilize fat as fuel. This suggests that during everyday training sessions aiming at enhancing mitochondrial capacity and endurance performance, carbohydrate intake in conjunction with conducting the training sessions may hamper the metabolic training stimuli. However, if lack of carbohydrate availability causes the training intensity or duration of the training session to be reduced the benefits of leaving out the carbohydrates may be lost. Therefore, recommendation for CHO fueling in conjunction with performing endurance exercise should be situation specific, as suggested by Impey et al. [29] according to their ‘fuel for the work required’ model.

VO_2max and 6 K TT performance were both improved after the intervention period. Despite observing a greater increase in mitochondrial proteins in CHO-PRO, we found no significant difference between groups in improvements in VO_2max and 6 K TT performance. These findings are in line with carbohydrate periodization studies that, consistently show augmented cell signaling, gene expression and training induced increases in oxidative enzyme activity and protein content, but fail to detect superior performance outcomes [29, 40].

A large number of short term studies have assessed the isolated effect of PRO supplementation on short-term endurance performance [41], showing either no or small benefits in relation to performance and recovery [41]. We have previously investigated the effect of consuming PRO-CHO before and after or during training sessions compared to isocaloric CHO in top-class orienteers [5] and elite cyclist [42] during strenuous training camps. Here, we observed improved performance in 4 K run TT, and lower CK elevation in orienteers receiving PRO-CHO [5], but no difference in peak power (10s Wingate test), five-min bike TT and markers of muscle damage in cyclist [42]. However, these findings are not comparable to the present study, as changes in short term performance in response to strenuous training are primarily an indicator of differences in acute muscle recovery rather than differences in training adaptations.

To our knowledge, this is the first long term training study to compare supplementation of CHO-PRO to isocaloric CHO. Previous studies, have compared intake of PRO supplementation to an isocaloric CHO beverage [14, 15, 17] or a < 1 g sugar pill [16], showing contradicting results in relation to VO_2max and performance tests. One study found PRO to elicit greater increases in VO_2max, 10 K TT and lean body mass [15], while others have found no effect [14, 17], or increased lean mass and a trend towards greater 5 K TT in placebo [16]. Hence, more research is needed to clarify whether PRO supplementation pre and post exercise may have beneficial effects on sports performance. Our primary aim was to detect differences in mitochondrial adaptations between PRO-CHO and CHO. Hence, the present study was not powered to detect differences in performance outcomes. In this regard, future research should use an extended study duration to allow for greater mitochondrial adaptations and may also benefit from using a performance test of longer duration, which more heavily relies on aerobic capacity and fat oxidation, than a 6 K TT that relies more on a combination of aerobic and glycolytic metabolism.

A number of limitations should be kept in mind when interpreting the results of the present study. First, to investigate the influence of protein supplementation on endurance training adaptations, subjects were given fast digestive whey PRO hydrolysate pre and post exercise. Consequently, whether the positive effects observed are a result of the pre and/or post exercise PRO supplementation alone or in synergy cannot be elucidated based on the present design. Secondly, since we did not include a control group consuming a non-caloric beverage pre exercise, we are unable to clarify whether differences in training adaptations are attributed to the inclusion of PRO or the exclusion of CHO pre-exercise. Also, the recommendation of intake of protein before and after endurance training may be applicable for a training situation, where subjects normally do not intake protein at least 2 h pre and post. Thirdly, We did not familiarize the subjects to the 6 K TT protocol due to time limitations. The subjects were trained runners and thereby familiar with the distance and their physical limitations. Nevertheless, we cannot rule out that part of the improvement from baseline to the midway test is related to a familiarization to the test protocol. Fourthly, we investigated trained runners (VO_2max > 60 ml O₂ min^{− 1} kg^{− 1}), therefore, we are unable to tell if our findings translate to populations with lower training status or other sports. Finally, we utilized biomarkers to assess changes in mitochondrial content and activity, which possess some limitations in regard to capturing changes in the mitochondrial reticulum [43].