Cystine/Glutamine Mixture Supplementation Attenuated Fatigue during Endurance Exercise in Healthy Young Men by Enhancing Fatty Acid Utilization

Exercise-induced fatigue is a multi-origin physical and mental phenomenon. Efforts to diminish the above predisposition may contribute to endurance, along with athletic well-being, while development of nutritional strategies to optimize condition and exercise performance are essential issues for athletes and trainers. Dietary amino acids are being discussed for their specific health-promoting properties beyond their role as building blocks of proteins. Glutamine, along with cysteine, are two kinds of amino acids that are reported extensively for their anti-oxidation, anti-inflammation, and immune-regulation properties, and are promising in sport applications. In the present study, we designed a randomized, placebo-controlled, crossover trial to examine effects of 7-day supplementation of cystine/glutamine mixture (Cys2/Gln) on self-reporting fatigue index (ratings of perceived exertion, RPE), energy metabolism, and inflammation. We also employed a C2C12 myotube model to examine the capacity of cystine for fatty acid utilization. Cys2/Gln supplementation alleviated fatigue by decreasing RPE and enhanced fatty acid oxidation during a 60 min endurance exercise in human trials, while cystine increased fatty acid utilization in C2C12 myotubes by enhancing mitochondrial respiration. In summary, Cys2/Gln supplementation exerts positive effects on ameliorating exercise-induced fatigue, mechanisms of which can be attributed to enhancement of fatty acid utilization.


Introduction
Fatigue is a symptom that can be associated with a state of exhaustion, following strenuous activity or physical exercise. At present, deletion of energy substances [1], accumulation of metabolites [2], production of reactive oxygen species (ROS) [3,4], and up-regulated inflammatory reaction [5] are all considered to be the factors that lead to fatigue. Fatty acid utilization capacity, including fatty acid mobilization and fatty acid oxidation (FAO), can also contribute to delay fatigue, and is deeply associated with exercise performance, especially endurance [6]. In addition to training, dietary intervention has been extensively applied to enhance exercise capacity based on these principles and hypotheses [7]. Specific nutrients, for instance, glutathione (GSH), are reported to enhance endurance by increasing FAO potential or quenching ROS [8]. Therefore, monitoring free fatty acid metabolism may be applicable for evaluating specific nutrients' potential toward FAO capacity.
Dietary amino acids are being discussed for their specific health-promoting properties beyond their roles as building blocks of proteins, including relieving fatigue and improving exercise performance [9]. Of common amino acids, cysteine and glutamine are garnering attention for their potential to improve exercise performance by ameliorating exercise-induced inflammation and oxidation, preserving gastrointestinal (GI) function,

Experimental Design and Experimental Protocol
We designed a double-blind, randomized, placebo-controlled crossover study. As shown in Figure 1, the participants were recruited to participate in two separate experimental trials with a 3-week wash out period: supplementation of either (1) Cystine/Glutamine mixture (Cys2/Gln) administration, the amino acid mixture trial (AAM trial) or (2) placebo protein trial (PLA trial). The whole experiment was carried out in a separated manner, characterized as Experiment 1 (Ex.1) and Experiment 2 (Ex.2). Protocols and flows of Ex.1 and Ex.2 were identical. After 5-day pre-exercise consumption, on the 6th and 7th day of each trial, participants were instructed to fast after 9:00 PM one night before and avoid water ingestion after 0:00 AM. Participants were then asked to urinate in the morning 2 h before arriving at the laboratory between 9:00 and 9:30 AM without consuming breakfast, urine and pre-exercise blood samples were then collected. Participants were then asked to consume the amino acid mixture or placebo with 200 mL water, rest in their seats for 30 min, and complete the questionnaire during the rest time. After a warm-up for 3 min at 60-90 W, the participants exercised on a cycle ergometer for 60 min at a target intensity of 70% targeted heart rate. Blood and urine were collected at the indicated time points (Figure 1). The experimental protocols were approved by the Ethics Committee of Waseda University (2016-288) and the Ethics Committee of Ajinomoto Co. . Written informed consent was obtained from all participants prior to their enrollment in the study. The experiments were carried out from September 2017-February 2018. This study was registered in advance using the University Hospital Medical Information Network Center, a system for registering clinical trials (ID: UMIN000026008). During the experiment, ratings of perceived exertion (RPE) were obtained using the Borg scale [25]. A questionnaire for evaluation of the physical condition (QPC) was finished on the final day of each trial. Fatigue/muscle soreness, gastrointestinal symptoms, cold symptoms, and insomnia symptoms were an-swered using a 7-point scale, where 1 point means "absolutely no" and 7 points means "absolutely yes". was registered in advance using the University Hospital Medical Information Network Center, a system for registering clinical trials (ID: UMIN000026008). During the experiment, ratings of perceived exertion (RPE) were obtained using the Borg scale [25]. A questionnaire for evaluation of the physical condition (QPC) was finished on the final day of each trial. Fatigue/muscle soreness, gastrointestinal symptoms, cold symptoms, and insomnia symptoms were answered using a 7-point scale, where 1 point means "absolutely no" and 7 points means "absolutely yes".

Participants and Amino Acid Mixture Supplementation
In total, 16 young men were recruited to the study, 13 of them finished this study. The participants were recreationally active and had no chronic diseases. Participants read and signed an informed consent form prior to engaging in the study. Inclusion criteria were as follows: (a) participants had to be male students from Waseda University between the ages of 20 and 40 years old, (b) participants had to be healthy and free of any known disease determined by a medical history questionnaire, (c) not allergic to soy protein, (d) participants had to avoid the performance intensive training during the experimental period, and Sports 2022, 10, 147 4 of 12 (e) participants had to abstain from non-steroidal anti-inflammatory drugs, gastrointestinal medicine, amino acid supplements, and alcohol for 3 days prior to participation. The amino acid mixture (2.46 g/pack, glutamine 1 g, cystine 0.23 g, and maltodextrin 1.23g in each pack) or the placebo (2.46 g/pack, maltodextrin 2.46 g in each pack) was consumed by the subjects 3 times per day (10:00 AM, 3:00 PM, and immediately before bedtime) for a consecutive 7 days during the experiment (Figure 1). On day 6 (1st exercise day) and day 7 (2nd exercise day), a consecutive two-day exercise experiment was carried out. The doses and regimens of glutamine and cystine were chosen based on previous studies that successfully observed a suppressive effect on oxidative stress without adverse effects in healthy adults [26][27][28].

Fatty Acid Oxidation Assay and Mitochondrial Oxygen Consumption Rate
After exposure of H 2 O 2 with/without Cys2 treatment, cells were washed twice with 1× KHB buffer (110 mM NaCl, 4.7 mM KCl, 2 mM MgSO 4 , and 1.2 mM Na 2 HPO 4 , pH 7.4, Sports 2022, 10, 147 5 of 12 2.5 mM glucose, and 0.5 mM carnitine) and incubated in a CO 2 -free incubator at 37 • C for 60 min. Following incubation, baseline measurements of the oxygen consumption rate (OCR) were recorded as an indicator of basal oxidative metabolism, using the Extracellular Flux Analyzers XFp (Agilent Technologies, Santa Clara, CA, USA). The palmitate-BSA conjugate was injected to a final concentration of 120 µM palmitate to measure the extracellular oxygen consumption rate for 80 min. The mitochondrial stress test was then performed to assess the bioenergetic status of the cells [26]. Cells were treated with oligomycin (a complex V inhibitor, final concentration 3 µM) at 20 min to induce maximal glycolytic metabolism, FCCP (carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone; uncoupling agent, final concentration 3 µM) at 50 min to uncouple electron transport and induce peak OCR, and antimycin A and rotenone (complex III and I inhibitors, respectively, final concentration 0.5 µM each) at 80 min to reveal non-mitochondrial respiration, after injection of the palmitate-BSA conjugate [29]. The maximal respiration after the addition of FCCP then reflects the maximal capacity of the electron transport chain [30]. The maximal respiration rate was calculated as the maximal OCR minus non-mitochondrial basal OCR determined after antimycin A and rotenone.

Sample Size Calculations and Statistical Analyses
The sample size was determined to be 16 based on our pilot study and a previous study [9]. The validity of this sample size was examined by calculating the statistical power. The sample size was validated using RPE as the primary study outcome, using the same sample size calculation methods used in a previous crossover study [9]. Analyses were performed using G*Power 3.1.0 (Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany) [31]. Due to withdrawals, the final sample size was 13. We validated the achieved power based on our data. The effect size was set to 0.8 based on a calculation using means. For two trials with an alpha level set at 0.05 and a correlation of 0.5, a total sample size of 13 would provide 91% power to detect between-trial differences. All values are expressed as mean ± standard deviation. For human trials, a linear mixed model for repeated measures (intervention, time (treated as categorical data), interaction (between intervention and time), and period related to a crossover study design as fixed factors and participants as the random factor) was applied. VO 2 during each trial was compared using a one-way repeated measures analysis of variance (ANOVA). A two-way repeated measures ANOVA was applied to determine the main effect of time and trial, and the interaction effect of time × trial on other variables. A linear mixed effect model was applied for the 2-trial comparison. For cell experiments, for comparison between the two groups, Sidak's multiple comparison test following an analysis of variance test was used. For comparison among three groups, significant differences were determined using Tukey's multiple comparisons test. Data were analyzed using SPSS Statistics, version 25 (SPSS Inc., Chicago, IL, USA) or GraphPad Prism 7.04 software (GraphPad Software Inc., San Diego, CA, USA), with p < 0.05 being considered significant. Part of data in free fatty acid analyses were treated as missing values by the analyzing software. For questionnaire analyses, there was no consideration for the multiplicity between evaluation outcomes, and between time points. All analyses in this study were planned and conducted after the unblinding for exploratory purposes.

A 7-Day Amino Acid Supplementation Decreased Ratings of Perceived Exertion
We employed the Borg Scale to evaluate RPE, an index of fatigue by self-reporting. RPE was measured every 5 min during each 60 min exercise test. As shown in Figure 2, on both days 6 and 7, after the exercise test started, participants in the AAM trial reported a lower RPE index at the timepoints of the 25th, 30th, 35th, 40th, 45th, 50th, and 55th minutes (p < 0.05). . RPE, ratings of perceived exertion. AAM trial, amino acid mixture trial. PLA trial, placebo trial. Pre, before exercise. Post, immediately after exercise. Post 30 min, 30 min after the exercise ends. Post 60 min, 60 min after the exercise ends. * p < 0.05. A linear mixed model for repeated measures (intervention, time (treated as categorical data), interaction (between intervention and time) and period related to a crossover study design as fixed factors and participants as the random factor) was applied for the two intervention comparison (total n = 13). . RPE, ratings of perceived exertion. AAM trial, amino acid mixture trial. PLA trial, placebo trial. Pre, before exercise. Post, immediately after exercise. Post 30 min, 30 min after the exercise ends. Post 60 min, 60 min after the exercise ends. * p < 0.05. A linear mixed model for repeated measures (intervention, time (treated as categorical data), interaction (between intervention and time) and period related to a crossover study design as fixed factors and participants as the random factor) was applied for the two intervention comparison (total n = 13).

A 7-Day Amino Acid Supplementation Enhanced Fatty Acid Utilization
We measured baseline and post-exercise plasma free fatty acid concentrations at several timepoints in both trials. As shown in Figure 3, on the 1st exercise day (day 6), immediately after exercise, plasma free fatty acid is significantly lower in the AAM trial than that in the PLA trial (p < 0.05). We also measured glycerol and ketone bodies, which are metabolites of triglyceride, but no significance was observed between trials (data was submitted as Supplementary Table S1).
We measured baseline and post-exercise plasma free fatty acid concentrations at several timepoints in both trials. As shown in Figure 3, on the 1st exercise day (day 6), immediately after exercise, plasma free fatty acid is significantly lower in the AAM trial than that in the PLA trial (p < 0.05). We also measured glycerol and ketone bodies, which are metabolites of triglyceride, but no significance was observed between trials (data was submitted as Supplementary Table S1). , interaction (between intervention and time), and period related to a crossover study design as fixed factors and participants as the random factor) was applied for the two intervention comparison (total n = 13).

Effects of Exercise and Amino Acid Supplementation on Blood Biochemistry and the Subjective Physical Condition
We measured various blood biochemistry markers in all trials. Plasma lactate, total protein, albumin, A/G, total bilirubin, AST, ALT, LDH, γ-GTP, CK, amylase, T-CHO, LDL-CHO, HDL-CHO, aldolase, triglyceride, uric acid, blood urea nitrogen, creatinine, glucose, bile acid, blood concentration of calcium ion, chloride ion, potassium ion, sodium ion, and inorganic phosphorus were not altered by exercise or amino acid supplementation (data are submitted as Supplementary Table S1). , interaction (between intervention and time), and period related to a crossover study design as fixed factors and participants as the random factor) was applied for the two intervention comparison (total n = 13).

Effects of Exercise and Amino Acid Supplementation on Blood Biochemistry and the Subjective Physical Condition
We measured various blood biochemistry markers in all trials. Plasma lactate, total protein, albumin, A/G, total bilirubin, AST, ALT, LDH, γ-GTP, CK, amylase, T-CHO, LDL-CHO, HDL-CHO, aldolase, triglyceride, uric acid, blood urea nitrogen, creatinine, glucose, bile acid, blood concentration of calcium ion, chloride ion, potassium ion, sodium ion, and inorganic phosphorus were not altered by exercise or amino acid supplementation (data are submitted as Supplementary Table S1).

Effect of Cys2 on Mitochondrial Respiration and Fatty Acid Utilization in C2C12 Myotubes
Following the injection of the palmitate-BSA conjugate, OCR significantly increased in the H 2 O 2 and H 2 O 2 + Cys2 groups compared with that of the control group (Figure 4a). Additionally, delta OCR significantly increased in the H 2 O 2 and H 2 O 2 + Cys2 groups compared with that in the control group (p < 0.01) (Figure 4b,c). Furthermore, the H 2 O 2 + Cys2 group was significantly increased compared with that of the H 2 O 2 group (p < 0.01, Figure 4b,c). Moreover, we measured the mitochondrial maximal respiration in the medium added with the palmitate-BSA conjugate. The H 2 O 2 group showed significantly decreased mitochondrial maximal respiration compared with that of the control group (p < 0.01), and the H 2 O 2 + Cys2 group showed a significant increase in mitochondrial maximal respiration compared with the H 2 O 2 group (p < 0.05. Figure 4d,e). group was significantly increased compared with that of the H2O2 group (p < 0.01, Figure  4b,c). Moreover, we measured the mitochondrial maximal respiration in the medium added with the palmitate-BSA conjugate. The H2O2 group showed significantly decreased mitochondrial maximal respiration compared with that of the control group (p < 0.01), and the H2O2 + Cys2 group showed a significant increase in mitochondrial maximal respiration compared with the H2O2 group (p < 0.05. Figure 4d,e). in delta OCR after injection of the palmitate-BSA conjugate, (c) delta OCR at 80 min after injection of palmitate-BSA conjugate, and (d,e) time-course changes in OCR and mitochondrial maximal respiration after injection of the palmitate-BSA conjugate. Data are shown as the mean ± standard error (n = 10-18/group). * p < 0.05; ** p < 0.01 (vs. control), # p < 0.05 (vs. H2O2). Con, incubation in 1/5 DMEM without H2O2 exposure; Oligo, oligomycin (a complex V inhibitor); FCCP, carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone; uncoupling agent); Rot/Ant, antimycin A and rotenone (complex III and I inhibitors, respectively).

Discussion
Exercise triggers various physiological responses in the body. Though regular exercise training favors the redox system, thus helping to maintain cellular homeostasis, such as by increasing the levels of anti-oxidative molecules, such as Sirtuin 1 (Sirt1), and endogenous histone deacetylase inhibitor, such as β-hydroxybutyrate, acute bouts of strenuous exercise may induce unfavored oxidative stress [32,33]. Therefore, anti-oxidative supplementations are being used extensively between athletes and sport-active populations.
Glutamine has been used as a sport supplementation for decades due to its outstanding anti-oxidation properties. Acute or chronic glutamine supplementations have been widely applied to take advantage of its antioxidant, anti-inflammatory, and immuno-regulating capacity. Recently, Caris and colleagues reported that 6-day glutamine (20 g/day) supplementation enhances in-trial glycemia and attenuates the increase in RPE (n = 9, treadmill exercise to exhaustion, 70% VO 2max ) [34]. Cystine is an oxidized form of cysteine. In 2018, a review summarized the potential health-promoting effects of cystine, that are supported by clinical evidence, including protective effects toward digestive diseases, the alleviating role in chronic inflammation by acting as a crucial antioxidant, controlling glycaemia, and vascular inflammation [35]. Fatty acids are important respiratory substrates during intensive cellular respiration activities, for instance, under the context of physical activity. Meanwhile, beta-oxidation, which is the main form of FAO, is in the mitochondrial matrix [36]. Enhancing the total capacity of FAO by increasing mitochondrial respiration had proved to be an applicable way to enhance exercise capacity [37]. In the present study, we discovered that Cys2/Gln supplementation showed an increasing effect on FAO, in human trials. To investigate whether and why cystine can enhance fatty acid utilization rate, we built a H 2 O 2 -activated C2C12 myotube model, which is employed as a model of an exercise-induced oxidative environment, the supplementation of cystine significantly enhanced mitochondria respiration and fatty acid uptake. To our knowledge, this is the first study demonstrated that cysteine/cystine is reported for its effects on enhancing fatty acid utilization energy production, therefore, the mechanism of cysteine/cystine's health-promoting capacities is unveiled further.
In other studies, it has been shown that cysteine supplementation can significantly reduce the activation of nuclear factor-kappa B (NF-κB) in activated human THP-1 (macrophage) cell lines [38], as well as significantly inhibit the expression of an intercellular adhesion molecule (ICAM)-1 [39] and production of IL-8 in both the THP-1 cell line and peripheral blood mononuclear cells (PBMCs) [38]. The authors also mentioned the possibility of amplifying the effects of cysteine by combining it with other nutrients, such as theanine, to achieve a maximal effect [40][41][42][43][44][45]. Previous research also pointed out that 10day supplementation of 700 mg cysteine combined with 280 mg of theanine supplementation ameliorated the inflammation response after a prolonged period of intense exercise (n = 15, an average total distance of 152.2 km), characterized by unaffected high-sensitivity C-reactive protein (hs-CRP) level, neutrophil count, and lymphocyte count, compared to the placebo takers [46]. However, we did not observe a significant anti-inflammatory or anti-oxidation effect of Cys2/Gln supplementation in our experimental model (data accessible as Supplementary Table S1), whether Cys2/Gln supplementation can exert positive effects in humans during exercise should be further studied using various experimental models to obtain convincing results.
Cysteine is also a building material of glutathione, while glutathione plays key roles in antioxidant defense, nutrition metabolism, regulation of cellular events, including gene expression, signal transduction, cytokine secretion, and immune response [47]. Homeostasis of glutathione has great implications for health, as well as athletic condition [45]. Endurance-exercise-induced lipid peroxidation can be minimized by glutathione supplementation [42], while glutathione supplementation is reported to suppress muscle fatigue induced by prolonged exercise in both animal and human models [48]. In the indicated study, the peroxisome-proliferator-activated receptor (PPAR)-γ coactivator-1α protein and mitochondrial DNA levels were significantly higher after glutathione supplementation (25% and 53% higher, respectively) in a murine model [48]. Meanwhile, the elevation of blood lactate was suppressed by glutathione supplementation (15% decreased compared to the placebo group). It is supposed that cystine supplementation may alter the over-consumption of glutathione during endurance exercise by increasing bioavailable cysteine, contributing to biosynthesis of glutathione, ensuring the capacity to quench free radicals, therefore, ameliorating fatigue and enhancing exercise performance and athletic well-being [46]. In addition, it is demonstrated that oxidative stress could alter membrane permeability and influence Ca 2+ homeostasis, therefore inducing mitochondrial DNA mutations, and damaging the mitochondrial respiratory chain [49], while the antioxidative properties of cysteine and glutamine, as well as their consequent GSH, are potential rescuers upon mitochondrial dysfunction triggered by oxidative stress during exercise.
We admit that the present study has limitations. Firstly, on the 1st exercise day (day 6), RPE is significantly reduced and FFA oxidation is significantly improved; while on the 2nd exercise day (day 7), the 2nd day of the consecutive 2 days of exercise, though an ameliorating trend is still observed, no significance is found, which is in accordance with the FFA profile, where no significant difference is observed. The anti-fatigue properties created by a 7-day supplementation may be not enough for repeated exercise, a longer period of taking the supplementation may receive more powerful effects toward exercises performed on consecutive days. A recent study also argues that the state of energy and fatigue might be independent, therefore, introduction of the measurement on both energy and fatigue using unipolar measurement tools may provide new insights [50]. Secondly, we did not manage to observe anti-inflammatory effects of Cys2/Gln supplementation, despite the anti-inflammatory properties of the above amino acids being reported extensively. Due to our limited sample size and non-completion by some subjects, an enlarged sample size may be satisfying.
In brief, in the present study, we adopted a 60 min exercise test to validate the multieffects of a 7-day Cys2/Gln supplementation during and post-endurance exercise. The combination of cysteine/glutamine exerts a more powerful capacity on mobilization and oxidation of free fatty acids, therefore, alleviating fatigue. Further studies are encouraged to employ maximal or submaximal exercise capacity tests to examine the potential roles and possible mechanisms of Cys2/Gln supplementation on endurance.

Conclusions
A 7-day Cys2/Gln supplementation showed alleviating effects on fatigue by reducing the self-reporting fatigue index in a randomized, placebo-controlled, crossover trial. By using a C2C12 cell model, we showed that the mechanism may be associated with increased fatty acid utilization by enhancing mitochondrial respiration in the muscle.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/sports10100147/s1, Table S1: Effects of Exercise and Amino Acid Supplementation on Blood Biochemistry Parameters.  Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.