The bioenergetics of athletic training and competition require readily available pools of energy-generating substrates to support the demands of skeletal muscle metabolism. Athletes involved in endurance-type sports (e.g., stage cycling, marathons, and triathlons) commonly maximize carbohydrate (CHO) availability through CHO loading to increase glycogen stores, whereas athletes involved in brief or intermittent high-intensity events (e.g., weightlifting, sprinting) target strategies to increase their capacity to use the phosphagen pathway, through creatine (Cr) supplementation.
Adenosine triphosphate (ATP) is the metabolic intermediary in the energy flow from stored energy substrates (e.g., Cr and glycogen) to muscular contraction as well as numerous other cellular processes (11). The primary role of intramuscular Cr is to rapidly rephosphorylate adenosine diphosphate to ATP for energy. Both dietary and endogenous Cr enters muscle cells via the insulin-sensitive SLC6A8 Cr transporter where ~60% is bound to a phosphate group and stored as phosphocreatine (PCr). The concentration of PCr in the muscle is threefold to fourfold greater than that of ATP but can be exhausted quickly during high-intensity resistance and sprint exercise. The coingestion of CHO with Cr potentiates an insulin-mediated increase in Cr transport, maximizing intramuscular Cr stores (8), which when combined with resistance-type exercise training has been shown to result in greater lean BM and strength.
There is evidence that Cr loading (20 g·d−1 for 5 d) in conjunction with a moderate-CHO diet (~6 g·kg−1 CHO for 3 d) results in a substantial (53%) increase in muscle glycogen content when compared with CHO consumption alone (18). Recent studies have also determined that enhanced glycogen capacity resulting from Cr supplementation (20 g·d−1) peaks within 24 h after exhaustive (70% V˙O2peak) exercise (24). This response is thought to be the result of increased cell size due to Cr- and CHO-induced water retention (12) and is associated with the up-regulation of signaling pathways mediating glycogen and protein synthesis, namely 5′ adenosine monophosphate–activated protein kinase (AMPK)– and mechanistic target of rapamycin (mTOR)–mediated signaling (12,25). However, it is presently unknown whether concomitant Cr and CHO loading strategies can enhance endurance exercise performance outcomes during the course of, rather than after, glycogen-limiting events (e.g., time trial (TT) cycling). Hence, the aim of this study was to investigate whether glycogen “super-compensation” with Cr and high-CHO intake leads to improved performance in well-trained cyclists during a laboratory protocol involving a 120-km TT and a simulated hill climb, with the latter element being included to investigate the effect of the increased BM expected as a result of the loading strategies. In light of previous research reporting water retention/changes in cell size as a result of Cr and CHO supplementation altering signaling pathways associated with protein and glycogen synthesis, we investigated proteins with putative roles in Cr and/or CHO metabolism to provide further insight into the molecular adaptations associated with the present exercise and dietary intervention.
METHODS
Subject
Eighteen endurance-trained male cyclists and triathletes with >2-yr racing history and currently cycling >250 km·wk−1 commenced the study with age, BM, maximum oxygen uptake (V˙O2peak), and peak power output (PPO; mean values ± SD) of 31.2 ± 5.8 yr, 78.2 ± 8.7 kg, 65.1 ± 7.1 mL·kg−1·min−1, and 388 ± 42 W, respectively (see Table, Supplemental Digital Content, Breakdown of subject characteristics (age, BM, V˙O2max, and PPO) for study inclusion presented as mean ± SD, https://links.lww.com/MSS/B16). Subjects who had taken any form of Cr supplement within 6 wk of the first PT or who had a history of abnormal bleeding, clotting, or seizure were excluded from the study. Experimental procedures and risks associated with the study were explained to all subjects who gave written informed consent before participating. The study was approved by the Human Research Ethics Committees at the Australian Catholic University (Reference No. 20140612) and the Australian Institute of Sport (Register No. 2014 254N). The study was registered with the World Health Organization’s International Clinical Trials Registry (UTN: U1111-1161-0890) and conducted in conformity with the policy statement regarding the use of human subjects according to the latest revision of the Declaration of Helsinki.
Study overview
On separate days after familiarization (described subsequently), subjects completed three cycling PT that consisted of a 120-km TT ride on an electromagnetically braked cycle ergometer immediately followed by a ride to volitional fatigue on an inclined treadmill set at a speed that elicited ~90% V˙O2peak. After the first baseline (standardized 6 g·kg−1 BM CHO diet) PT, subjects were pair matched into Cr- or PLA-supplemented groups on the basis of PPO, performance measures from PT1, and dual-energy x-ray absorptiometry lean BM measures in a double-blinded allocation. All subjects then undertook a randomized crossover application of the CHO intervention, consuming either a moderate (6 g·kg−1 BM·d−1; MOD) or CHO-loaded (12 g·kg−1 BM·d−1; LOAD) diet 2 d before PT2 or PT3. A total of four muscle biopsies were obtained before PT1, 18 h after PT1 (i.e., glycogen depleted), and before PT2 and PT3 for biochemical analysis of intramuscular metabolites.
Preliminary testing
Upon arrival to the laboratory and after voiding, a nude measure of BM was obtained for V˙O2peak and energy intake calculations. Subjects were subsequently weighed in full racing kit (e.g., jersey, cleats, knicks, socks, and helmet) and bicycle to calculate a relative treadmill speed for the hill climb simulation (described subsequently). The V˙O2peak and PPO of each subject were determined using an incremental test to volitional fatigue on an electromagnetically braked cycle ergometer (Lode, Groningen, the Netherlands). The test protocol commenced at 150 W for 5 min and progressed to 250 W. After 150 s, work load increased by 50 W, with all subsequent work load increasing by 25 W every 150 s until volitional fatigue, which was determined by the inability to maintain cadence >70 rpm. PPO was determined to be the power output of the highest stage completed plus the fraction of any uncompleted workload (W). Expired gasses were collected into a calibrated customized Douglas bag gas analysis system, which incorporated an automated piston that allowed the concentrations of O2 and CO2 and the volume of air displaced to be quantified. V˙O2peak was calculated as the highest average O2 consumption recorded for 60 s.
After the V˙O2peak test, subjects undertook a 60-km familiarization TT on a cycle ergometer (Velotron, Seattle, WA) with 1- and 4-km sprint efforts (e.g., “as fast as possible”) alternating every 10 km, during which time they consumed 60 g of CHO per hour in the form of a sports drink or gel. Upon the completion of the TT, subjects performed a timed ride to exhaustion (criteria subsequently defined) on a customized treadmill (Australian Institute of Sport, Canberra, Australia) set at an 8% gradient at a speed that elicited ~90% of a subject’s V˙O2peak as previously established (5). This intensity was chosen to mimic hill climb durations (~10–30 min) commonly observed in international road cycling races. The first study PT commenced within 7 d of familiarization.
Study diet and exercise protocol
Dietary control was implemented for the 2 d before each PT using a prepackaged standardized diet protocol. An individualized menu was constructed for each subject using FoodWorks Professional Edition, Version 7.0 (Xyris Software, Brisbane, Australia) on the basis of their BM and food preferences. Subjects received a moderate-CHO (MOD) diet providing 6 g·kg−1 BM·d−1 CHO, 1.5 g·kg−1 BM·d−1 protein, 1.5 g·kg−1 BM·d−1 fat, with a total energy of ~215 kJ·kg−1 BM·d−1 2 d before undertaking their first baseline PT (Fig. 1). Subjects refrained from any intake of alcohol during the dietary standardization period. Caffeine intake was allowed ad libitum up to 2 d before each PT and up to 2 standard servings (e.g., one cup of coffee or one can of caffeinated soft drink) the day before the experimental trial. Subjects recorded their caffeine intake, and this was repeated during the dietary standardization period of subsequent PT. Subjects were provided with all food and drinks in their standardized menu in portion-controlled packages and were given verbal and written instructions on how to follow the diet. Checklists were used to record each menu item as it was consumed and to note any deviations from the menu. On arrival at the laboratory for each PT, this checklist was cross-checked for compliance with study requirements. Hydration status was assessed using the specific gravity test (UG-a; Atago Refractometer, Tokyo, Japan) on an “on-waking” urine sample.
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FIGURE 1: Study design and timeline.
On the morning of each PT, subjects reported to the laboratory at the same time (0700–0800 h) after an overnight fast. After 20 min of rest in a supine position, a muscle biopsy was obtained under local anesthetic (2–3 mL of 1% xylocaine) from the vastus lateralis using a 5-mm Bergstrom needle modified for manual suction (2). Muscle samples were immediately snap-frozen in liquid N2 and stored at −80°C until later analysis. A standardized “prerace” meal providing 2 g·kg−1 BM CHO was consumed 2 h before the start of each PT. Subjects were asked to void before the start of the PT to obtain a starting weight and began the PT exactly 2 h after breakfast.
PT consisted of a 120-km self-paced cycling TT on a cycle ergometer during which subjects performed maximal intermittent high-intensity sprint efforts alternating between 1 and 4 km every 10 km (Fig. 1). Power output was measured as an average of all data points collected from the start to the end of each individual 1- and 4-km effort as recorded by the cycle ergometer software (Velotron CS 2008) as well as an overall average of 1- or 4-km sprints comparatively. A fan that maintained air circulation (15–17 m·s−1) and cooling was positioned 3 m away from the subject for all trials. Heart rate readings (RS300; Polar Electro, Kempele, Finland) were obtained both before and after the completion of each sprint. Rating of perceived exertion (RPE; Borg scale) was obtained at the completion of each sprint effort. Subjects were required to follow a standardized hydration and CHO intake (60 g·h−1) plan, which was recorded and repeated in subsequent PT. Upon the completion of the TT, subjects quickly dismounted the ergometer where they voided, toweled off, and were reweighed. Subjects then rode their own bicycle on an inclined treadmill set at an 8% gradient at a speed that elicited ~90% of a subject’s V˙O2peak. Subjects rode until volitional fatigue, which was determined to be three verbal warnings that they had drifted back beyond a predetermined safety zone on the treadmill or when subjects grabbed onto the side of the treadmill safety bar. The transition from ergometer to treadmill was <5 min. Subjects received similar encouragement through all PT and did not receive any feedback on their performance until the completion of the study.
After the completion of the first PT, subjects were fed a prepackaged standardized low-CHO diet (<1 g·kg−1 BM) for the remainder of the day to minimize resynthesis of muscle glycogen stores (4). Participants then reported to the laboratory in a fasted state the next morning (“glycogen depleted”) where a second muscle biopsy was taken under rested and fasted conditions. After the biopsy, subjects were randomized into either Cr-loaded (20 g·d−1 for 5 d followed by 3 g·d−1 until the end of the trial) or PLA cohorts (Fig. 1) using a pair-matched design on the basis of PPO (W·kg−1), results of the first PT, and dual-energy x-ray absorptiometry lean mass estimates. In the 2 d before each of the two additional PT (commenced 1 wk apart), subjects receive either a repeat of the MOD diet or a CHO-loaded (LOAD) diet (12 g·kg−1 BM·d−1) in a crossover allocation (Fig. 1). These dietary treatments were implemented using a PLA-controlled design whereby the overall menu for the day was kept constant, but key items were provided either as a low-energy/low-CHO option or an indistinguishable high-energy/CHO-enriched form. Protein and fat intake each remained constant at 1.5 g·kg−1·d−1 in these diets, but energy intake was increased in the CHO LOAD diet (~320 kJ·kg−1·d−1). Subjects completed all subsequent biopsies and PT as described; however, no dietary restriction or follow-up biopsy was given after the second and third PT. Analysis of all the diets consumed (Foodworks) by participants was undertaken on completion of the study by a registered dietitian.
Determination of Cr content
Muscle Cr content was measured as described previously (10). Briefly, 20–30 mg wet weight muscle tissue was freeze-dried, weighed, and extracted with 1 M perchloric acid. Samples were analyzed in duplicate for free Cr, PCr, and ATP using fluorimetry. Total Cr was measured as a sum of free Cr and PCr.
Determination of glycogen content
Muscle glycogen content was measured as described previously (5). Briefly, ~20 mg of muscle tissue was freeze-dried and powdered with all blood and connective tissue removed. The freeze dried samples were extracted with 500 μL of 2 M HCl, heated to 100°C for 2 h to hydrolyze the glycogen to glycosyl units, and then neutralized with 1.5 mL of 0.67 M sodium hydroxide. Glycogen concentrations were determined via enzymatic analysis with fluorometric detection (Jasco FP-750 spectrofluorometer, Easton, MD) at excitation 365 nm/emission 455 nm.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting analysis
Quantification of protein expression was carried out as previously described (5). Approximately 20 mg of skeletal muscle was homogenized using a motorized pellet pestle in an ice-cold buffer containing 50 mM Tris–HCl (pH 7.5), 1 mM EDTA, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 50 mM NaF, 5 mM sodium pyruvate, 1 mM dithiothreitol, 10 μg·mL−1 trypsin inhibitor, 20 μg·mL−1 aproptinin, 1 mM benzmidine, and 1 mM phenylmethylsulfonyl fluoride. Samples were spun at 16,000g for 30 min at 4°C with supernatant collected for analysis. Total protein concentration was determined using a BCA protein assay (Pierce Biotechnology, Rockford, IL), with lysate resuspended in Laemmli sample buffer, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and transferred to polyvinylidene fluoride membranes blocked with 5% nonfat milk and washed with 10 mM Tris–HCl, 100 mM NaCl, and 0.02% Tween 20, and then incubated with primary antibody (1/1000) overnight at 4°C on a shaker. Membranes were incubated with secondary antibody (1/2000), and proteins were detected via enhanced chemiluminescence (Amersham Biosciences, Buckinghamshire, United Kingdom; Pierce Biotechnology) and quantified by densitometry (Chemidoc, BioRad, Gladesville, Australia). All samples for each individual were run on the same gel. Antibodies for total expression of peroxisome proliferator–activated receptor gamma coactivator 1-alpha (PGC-1α; no. 2187), mTOR (no. 2972), and 5′ AMPK (no. 2532) were from Cell Signaling Technology (Danvers, MA). SLC6A8 (no. AV42248) Cr transporter was from Sigma-Aldrich (St. Louis, MO). For all proteins, the volume density of each target band was normalized to the total protein loaded into each lane using stain-free technology (8).
Statistical analysis
Data were analyzed with the SPSS software package (version 22) using a general linear mixed model with repeated measures where the within-subjects variable was CHO condition (baseline, moderate, loaded) and the between-subject factor was treatment (PLA or Cr). All data were checked for sphericity using the Mauchly test, and where a significant difference was detected, the Greenhouse–Geisser test was used. Data were checked for normality using the Shapiro–Wilk test. When this test of normality failed, data were naturally log transformed before further analyses. All data in text and figures are presented as mean ± SD, with P < 0.05 indicating statistical significance.
RESULTS
Dietary compliance
Assessment of individual records revealed universal (100%) compliance with the dietary protocols. Cross-checked intakes of energy, CHO, protein, and fat intakes were similar to the prescribed diets, and there were no differences in nutrient intakes for each dietary protocol between intervention groups or between dietary protocols that were repeated within the same group (e.g., MOD diet, or prerace diet).
Changes in BM
Within the CR cohort, BM increased with LOAD above baseline (+1.49%; P < 0.001) and MOD (+1.36%; P < 0.001). Within the PLA group, BM was reduced below baseline when subjects consumed MOD (−0.78%; P < 0.05) but increased above baseline when they consumed LOAD (+1.54%; P < 0.001). LOAD also increased BM above MOD (+1.36%; P < 0.05) in the PLA group. LOAD resulted in an overall increase in BM above both baseline (+1.12%; P < 0.001) and MOD (+1.45%; P < 0.001) diets irrespective of CR/PLA treatments (Fig. 2A). No significant differences were observed between CR and PLA cohorts.
FIGURE 2: Change in BM between treatments. (A) Change in BM relative to baseline measures for all dietary intervention groups before the commencement of the TT portion of the PT (i.e., before any exercise). (B) Change in BM relative to baseline measures for all dietary intervention groups before the commencement of the hill climb portion of the PT. Values are expressed as percent change relative to baseline (dotted line) ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, denoting significant difference to baseline; #P < 0.001, denoting significant difference to CHO treatment (MOD vs LOAD) within the supplement.
In BM obtained just before commencement of the hill climb portion of the PT, differences between the CR and PLA groups were no longer significant (Fig. 2B). Within the CR cohort, BM increased with LOAD above baseline (+1.49%; P < 0.001) and MOD (+1.34%; P < 0.001). Within the PLA group, BM increased with the LOAD diet above baseline (+0.72%; P < 0.05) and MOD (+1.40%; P < 0.001) diets. LOAD resulted in an overall increase in BM above both baseline (+1.11; P < 0.001) and MOD (+1.37%; P < 0.001) diets irrespective of CR/PLA treatments (Fig. 2B). There was no significant difference in total weight lost between pre-TT and pre–hill climb weights (i.e., sweat loss) between the different trial dates or treatment interventions.
PT outcomes
There were no differences in 120-km TT time or between treadmill cycling time to exhaustion between trials (CHO diets or supplement; Table 1).
TABLE 1: Summary of cycling TT and treadmill performance–associated measures.
Power output and aerobic capacity
Within the PLA cohort, greater power and maximal aerobic power (MAP) were achieved, with both MOD (P < 0.05) and LOAD (P < 0.05) significantly increasing above baseline (Table 1). Within the 1-km sprint efforts, LOAD improved MAP above baseline (P < 0.05) during the final sprint effort in the PLA-treated group (Fig. 3C). Within the 4-km sprint efforts, CR with both MOD and LOAD significantly increased (P < 0.05) MAP above baseline during the final sprint effort (Fig. 3D). There were no differences in mean power output or percentage of MAP between the supplemented cohorts (Table 1).
FIGURE 3: Differences in 1-km (A) and 4-km (B) sprint effort power output (W) between treatments and relative to baseline (C and D for 1- and 4-km sprints, respectively). Data are presented as mean ± SD. #P < 0.05, denoting significance relative to baseline at that sprint interval.
RPE
There was a significant interaction between CHO diets and supplementation on RPE (P < 0.001). CR elicited a greater overall perceived exertion (16 vs 15; P < 0.001) compared with PLA. There was also a significant difference in perceived exertion between LOAD and baseline (P < 0.001). LOAD decreased RPE (P < 0.001) from baseline with CR. CR subjects reported significantly greater RPE when on both MOD (P < 0.001) and LOAD (P < 0.001).
Muscle glycogen
Muscle glycogen concentrations were significantly decreased (P < 0.05) after the first PT (“depleted”; 264 ± 102 mmol·kg−1 dm (dry mass)) compared with baseline (584 ± 135 mmol·kg−1 dm), MOD (579 ± 123 mmol·kg−1 dm), and LOAD (704 ± 111 mmol·kg−1 dm) irrespective of supplemented cohorts. The ingestion of LOAD resulted in greater muscle glycogen concentrations than that under baseline, depleted, and MOD conditions (P < 0.05). There were no differences in glycogen contents between baseline and MOD or between CR and PLA conditions (Fig. 4A).
FIGURE 4: Analysis of muscle metabolites. A, Skeletal muscle glycogen concentrations at baseline (CHO, 6 g·kg−1 BM·d−1), after PT (depleted), and after moderate CHO intake (6 g·kg−1 BM·d−1) and CHO Loading (12 g·kg−1 BM·d−1) in groups who undertook Cr loading or PLA. Values are presented as a mean ± SD. *P < 0.05, denoting significant difference between CHO treatments irrespective of supplement groups. B, Total Cr content in the muscle at baseline, after PT (depleted), and as a result of Cr loading (5 × 20 g·d−1; day 7) and Cr maintenance (3 g·d−1; day 14). C, Free and total Cr, PCr, and ATP data at baseline and after MOD CHO intake and CHO LOAD, noting that MOD and LOAD days were crossed over between day 7 and day 14. Values are presented as a mean ± SD. *P < 0.05, denoting significant difference from baseline. #P < 0.05, denoting significant difference between supplement groups at the CHO intervention.
Muscle Cr and ATP concentrations
CR supplementation increased total Cr above PLA by day 7 (P = 0.059), but this was not sustained by the maintenance dose at day 14 (Fig. 4B). CR-loaded subjects also had an increase in total Cr above PLA when CHO loaded (P = 0.053; Fig. 4C). Subsequently, values for total Cr and PCr at MOD and LOAD in the Cr group were higher than those at baseline for the CR-supplemented group and higher than the PLA group, with a significant change from baseline observed in the Cr-loaded cohort when on the LOAD diet (P < 0.05; Fig. 4C).
Protein analysis
The expression of total mTOR significantly decreased after glycogen depletion compared with baseline (~30%; P < 0.05; Fig. 5A). No significant changes were observed with total AMPK, although a trend toward reduced expression was observed after glycogen depletion with CR relative to baseline (Fig. 5B). No changes were observed in PGC-1α (Fig. 5C) and SLC6A8 (Fig. 5D) total protein expression.
FIGURE 5: Total mTOR (A), total AMPK (B), total PGC-1α (C), and SLC6A8 Cr transporter protein (D) in skeletal muscle taken before TT completed under baseline, glycogen depleted, moderate-CHO (MOD), or CHO-loaded (LOAD) diets with or without Cr supplementation. Images are representative blots, and values are expressed relative to total protein and presented in arbitrary units ± SD (n = 9 per group). *P < 0.05, denoting significant difference where indicated.
DISCUSSION
CHO loading is a common tactic used by endurance athletes as a means of increasing intramuscular glycogen stores, with research indicating that Cr and CHO coingestion can increase muscle glycogen storage, which may enhanced endurance-based exercise tasks (18,34). We report for the first time significantly greater power output during repeated high-intensity sprint efforts undertaken in the late stages (i.e., the late-stage sprints of a prolonged bout of endurance cycling when glycogen is depleted) of a simulated 120-km TT when cyclists combined Cr and CHO loading. We also report that this response was independent of muscle glycogen concentrations and total mTOR expression. Our results suggest that Cr, when coingested with CHO, may have a beneficial effect on specific in-competition aspects of endurance cycling performance, which mimicked the demands typically observed in multistage tours (i.e., multiple sprints and hill climbing) and typically results in glycogen depletion (1). The changes in power output we observed during sprints are likely to have a major practical effect on the final outcome of a race, because both cycling and running events are often won by the athlete who can either stay with the leading pack during breakaways or sprint to the finish line in the latter stages of a race. Although we did not detect a significant difference in overall performance times as a result of either Cr and/or CHO loading (perhaps due to variations in subject TT pacing strategies), an increase in pre-TT BM (Fig. 2A) due to these strategies did not negatively affect the weight-sensitive “hill climbing” simulation included at the end of our PT. This suggests that an avoidance of Cr use in endurance cycling, due to weight gain and potentially slower result times, may be unfounded.
The estimates of muscle Cr concentrations in our study suggest that the loading protocol was effective in increasing muscle Cr above baseline values; however, there is some uncertainty about the efficacy of the maintenance dose in sustaining these elevations in muscle Cr content. This may be an artifact of the variability of the technique (biopsy sampling plus the coefficient of variation of the Cr assays) or an indication that a Cr dose of 3 g·d−1 is insufficient, at least in the short period after high Cr doses, or maintaining such elevations (33). An inability to maintain elevated Cr concentrations may explain a lack of change in glycogen content between the CR and PLA groups and furthermore be a factor contributing to a lack of change in performance times. Studies have shown that Cr transporter activity can be regulated/enhanced by extracellular Cr concentrations (15) and the presence of insulin (28,31). We observed little further difference between groups in terms SLC6A8 protein expression; however, this is in line with previous studies on both short- and long-term Cr supplementation (0.125 g·kg−1·d−1 either 2 or 4 months), which showed no change in SLC6A8 mRNA content when combined with a standardized cycle exercise program (30). It is also possible that our exogenous loading dose down-regulated endogenous Cr production to such a large extent that there was an undershoot in total muscle Cr in the phase immediately after the cessation of this dose (27,33). Regardless, because we followed a protocol that is evidence based and showed numerical increases in muscle Cr content at both loading (P = 0.059) and maintenance phases (LOAD day; P = 0.053), we feel confident that the Cr-supplemented group achieved muscle Cr content at both the MOD PT and at LOAD PT that was functionally higher than that in their baseline trial and in comparison with the PLA group.
Cr’s ergogenic benefits stem from its ability to act as a “reservoir” for substrates that are required for the rapid regeneration of energy during brief periods of high-intensity, maximal effort. In contrast to previous work that showed that Cr did not increase power output during short (200 m) sprint efforts during a 25.2-km cycling sprint trial (13), we report that Cr loading increases power output during the late stages of maximal sprint efforts performed during a 120-km cycling TT lasting ~3 h. Work by Vandebuerie and colleagues (32) reported a ~9% increase in power output in sprint efforts (5 × 10 s) of Cr-loaded (25 g·d−1 for 5 d) elite cyclists only after a 2.5-h TT cycling protocol. Oliver and colleagues (19) subsequently demonstrated that a Cr loading protocol (20 g·d−1 for 6 d) further supplemented with glucose (15 g·d−1) was able to reduce lactate concentrations during exercise but with no difference in maximal power output or total time to fatigue during incremental cycling. Our study is the first to show a trend toward increased sprint power with Cr during the course of, rather than after, a prolonged endurance cycling bout. Although the Cr and/or CHO loading did not provide a detectable benefit to overall performance of the 120-km cycling TT, we note that the increase in pre-TT BM associated with the single and combined use of these strategies did not impair capacity for a weight-sensitive exercise element in our cycling protocol (wherein no significant change in BM was identified because of hydration strategies being kept constant between all PT). This latter finding is of practical significance because many endurance athletes are disinclined to use Cr as a performance supplement because of fears that the associated BM gain will impair performance in events where power-to-mass ratios are important.
Although it is likely that observed increase in power output is due to the ability of Cr to rapidly rephosphorylate ATP for rapid energy generation, recent evidence indicates that this response may also be a result of decreased muscle glycogen utilization and muscle protein degradation (29). Specifically, Tang and colleagues (29) observed consistent decreased lactate levels indicative of decreased muscle glycogenolysis in subjects supplemented with Cr (12 g·d−1 for 15 d) after an endurance exercise session involving a 60-min run followed by 100-m sprint efforts. Lactate measures obtained from our subjects after the sprint bouts tended to be lowered in the Cr-loaded cohort (data not shown), which may suggest “glycogen sparing,” but direct measures would be needed to confirm such a notion.
The addition of CHO to a traditional Cr loading strategy (20 g·d−1 for 3–5 d) has previously been reported to increase muscle glycogen stores (18,34). The basis for this response has centered around the idea that greater intracellular water retention is associated with increased Cr transport, which subsequently increases cell volume, promoting a greater capacity for glycogen storage (12,22,25,34,37). Nelson and colleagues (18) showed that subjects who consumed a moderate CHO (~6.6 g·kg−1 BM) diet after traditional Cr loading increased muscle glycogen content by 53% above those consuming high CHO alone. Similarly, van Loon and colleagues (33) demonstrated that 7 wk of prolonged Cr supplementation (20 g·d−1 for 5 d followed by 6 wk of 2 g·d−1) resulted in an 18% increase in muscle glycogen content. Neither of these studies, however, involved any exercise modality to test the implications or benefits of elevated glycogen stores on training and performance. We found that CHO loading increased muscle glycogen by ~17% above baseline, but observed no further increase in muscle glycogen contents or performance time when CHO loading was combined with Cr loading. Previous work by our group had failed to observe a change in a similar cycling protocol (100 km with 1- and 4-km sprint efforts) after CHO loading alone, suggesting that CHO consumed during exercise, such the subjects of the present study did, offsets any detrimental performance on lower preexercise glycogen concentrations (3). This may further be explained by recent findings indicating that the optimal window for increasing glycogen stores using a concomitant Cr and CHO supplementation strategy occurs within a narrow time frame (24). Roberts and colleagues (24) reported that 5 d of Cr supplementation (20 g·d−1) and high CHO (37.5 kcal·kg−1 BM·d−1) intake after an exhaustive bout of exercise (70% V˙O2max) resulted in augmented muscle glycogen contents within 24 h (↑ ~82% compared with PLA) with no subsequent changes in glycogen contents observed between Cr and PLA cohorts. These data, however, represent the potent effects of Cr and CHO loading on recovery rather than its effects on performance.
Although it is possible to conclude that glycogen is not important or limiting for this cycling performance under the circumstances of our study, which follow sports nutrition recommendations of consuming large amounts of CHO during exercise to support high-CHO availability (4), we also note that our ambitious protocol involved a number of performance parameters within the overall cycling test (i.e., sprint intervals spaced exactly 10 km apart, alternating 1- and 4-km sprints, the hill climb follow-up), and in hindsight, different pacing strategies used by the riders may have masked our ability to see an overall effect. Specifically, whether riders concentrated their efforts on the various sprints, the overall 120-km TT, or the hill portion of the protocol and whether this changed with supplementation and/or experience (despite our familiarization trial) could not be ascertained from the present study and provides a limitation to the final findings. In hindsight, a more straightforward protocol might have been more suited to finding a detectable change in performance even if it fails to replicate the complexity of real-life sport.
Cr induced a significant increase in BM compared with PLA, indicating that our ingestion protocol promoted additional intracellular water storage. Cell swelling caused by Cr and CHO loading via the storage of additional water within the muscle cells has been associated with the up-regulation of a large number of signaling markers involved in protein and glycogen synthesis (12,22,25,37). It has been hypothesized that Cr-mediated cell swelling may further result in the increased expression of key proteins involved in hypertrophy-related signal transduction (7), namely, mTOR (14). However, no studies have directly examined cell signaling responses when performing glycogen-depleting exercise (i.e., TT cycling/sprints to exhaustion) in concert with concomitant Cr and CHO loading.
Several lines of evidence indicate little involvement of Cr in myofibrillar and sarcoplasmic protein turnover both after resistance type exercise and at rest (16,17). We observed a decrease in total mTOR abundance in biopsies taken 18 h after a glycogen-depleting TT and exhaustive hill climb. However, in contrast to our hypothesis, mTOR expression was similar irrespective to whether participants were Cr and/or CHO loaded (Fig. 5). Although this indicates that Cr supplementation exerts no further beneficial effect on mTOR signaling, this finding further supports our glycogen data between conditions by demonstrating that Cr was unable to increase cell size through mTOR-mediated mechanisms to enhance glycogen storage capacity. mTOR has also been implicated in stimulating SLC6A8 Cr transporter via the serum and glucocorticoid-inducible kinase (SGK1), which is also involved in the regulation of cell volume and activity of various Na+/K+ carriers such as SLC6A8 (26). It is plausible that the decreased mTOR expression observed in our study attenuated activation of SGK1 resulting in submaximal transport of Cr into muscle and therefore negating any increase in cell volume and glycogen storage.
Endurance athletes benefit from a greater mitochondrial capacity because of their increased requirement for available energy. The activation of 5′ AMPK monitors metabolic and energetic states in muscle, which is affected by exercise stimuli (35) and is furthermore modulated by the PCr/Cr ratio (21). Our data showed no change in AMPK status despite observing a slight (~5%) drop in the PCr/Cr ratio after a glycogen-depleting PT. Other studies have reported that muscle with augmented glycogen content tended to have lower resting levels of AMPK and were also less sensitive to activation in response to stimuli such as exercise (23,36). Our failure to detect changes in AMPK expression may be because our subjects had adequate CHO (6 or 12 g·kg−1 BM CHO·d−1) in their diet 2 d before the completion of the first (and subsequent) PT as well as the fact that they consumed 60 g·h−1 of CHO during the ride itself. We also observed no change in PGC-1α, a signaling target of AMPK. A limitation to the present study was the time at which our biopsies were taken, which may have been too late to observe any changes in PGC-1α signaling as a result of our nutrient or exercise interventions (20). Future studies should investigate long-term Cr and CHO loading strategies with muscle samples obtained both before and immediately after bouts of exercise to further elucidate any beneficial adaptations.
In summary, we present novel data to demonstrate that the combination of Cr and CHO loading can increase power output during repeated high-intensity sprint efforts undertaken during late stages of prolonged simulated TT cycling, which mimicked the physiological demands typically observed in multistage cycle tours (i.e., multiple sprints and hill climbing). The increased power outputs were independent of muscle glycogen content. Although Cr loading tended to increase BM at the start of the PT, these differences became less significant over the course of the TT and, despite being heavier, did not interfere with performance of the weight-sensitive part of the TT (the hill climb). In fact, if anything, the performances were longer (better endurance) with the loaded conditions. Because cycling and running events are often won by the athlete who can either stay with the leading pack during breakaways or sprint to the finish line in the latter stages of a race, it is likely that the higher-power outputs observed during these late intense sprints would have a major effect on the final outcome of such races.
This study was funded by an Australian Catholic University Research Funding grant (2013000443) awarded to L. M. B.
The authors declare no conflict of interest. The results of the study are presented clearly, honestly, and without fabrication, falsification, or inappropriate data manipulation. The results of the present study do not constitute endorsement by the American College of Sports Medicine.
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