Microbiota Mediate Enhanced Exercise Capacity Induced by Exercise Training

Abstract

The gut microbiota is critical to host metabolism and is influenced by many factors including host genotype, diet, and exercise training.

Purpose:

We investigated the effects of gut microbes, and the mechanisms mediating the enhanced exercise performance induced by exercise training, i.e., skeletal muscle blood flow, and mitochondrial biogenesis and oxidative function in male mice.

Methods:

All mice received a graded exercise test prior to (PRE) and following exercise training via forced treadmill running at 60–70% of maximal running capacity 5 d/wk for 5 weeks (POST). To examine the role of the gut microbes, the graded exercise was repeated after 7 days of access to antibiotic treated water (ABX), used to eliminate gut microbes. Peripheral blood flow, mitochondrial oxidative capacity, and markers of mitochondrial biogenesis were collected at each time point.

Results:

Exercise training led to increases of 60 ± 13% in maximal running distance and 63 ± 11% work to exhaustion (p<0.001). These increases were abolished following ABX (p<0.001). Exercise training increased hindlimb blood flow and markers of mitochondrial biogenesis and oxidative function, including AMPK, sirtuin-1, PGC-1α citrate synthase, complex IV, and nitric oxide; all of which were also abolished by ABX treatment.

Conclusions:

Our results support the concept that gut microbiota mediate enhanced exercise capacity following exercise training and the mechanisms responsible, i.e., hindlimb blood flow, mitochondrial biogenesis, and metabolic profile. Finally, results of this study emphasize the need to fully examine the impact of prescribing ABX to athletes during their training regimens and how this may affect their performance.

INTRODUCTION

The gut microbiome is comprised of trillions of microorganisms and plays a crucial role in host metabolism, immunity, and several diseases (1–3). Several groups, including our lab (4), have examined the link between the gut microbiota and exercise (5–7). Exercise is not only central to healthful aging but has also been shown to improve the diversity of microbes within the Firmicutes phylum (4, 8, 9), and increasing the abundance of beneficial bacteria such as Roseburia intestinalis, Faecalibacterium prausnitzii, and Akkermansia muciniphila (10, 11).

To examine the impact of the microbiome on host physiology, studies typically employ the use of either germ-free models or antibiotic treatment (ABX) regimens to evaluate changes in host physiology (12). In 2015, Hsu et al. investigated the association of intestinal bacteria and exercise performance in specific pathogen-free, germ-free, and gnotobiotic mice, a group of mice given a known microbe, Bacteroides fragilis (7). Their group showed that endurance swimming time was significantly longer for specific pathogen-free mice compared with both Bacteroides fragilis and germ-free mice. In 2019, Huang et al. also showed that baseline fitness in untrained germ-free mice was significantly lower than the untrained specific pathogen-free mice. Importantly, the authors noted while germ-free mice could benefit from exercise training adaptations, it was still significantly lower than specific pathogen free groups (13). Taken together, an intact microbiota appears to be essential for exercise endurance and adaptations to exercise training. However, while the importance of the gut microbiota in regulation of exercise training are beginning to be established, it is not known how ABX administration during a training regimen can impact exercise performance.

While the overall influence of exercise on the gut microbiome is becoming more defined, the reverse knowledge is lacking, i.e., what is the impact of microbiota depletion on enhanced exercise performance following exercise training and the mechanisms mediating the enhanced exercise performance. Therefore, the purpose of the current investigation was to examine how a loss of gut microbes, via ad libitum access to ABX treated water, influenced enhanced exercise capacity following exercise training and the mechanisms mediating the enhanced exercise capacity. These mechanisms include mitochondrial function, mitochondrial biogenesis, and hindlimb blood flow. We hypothesized that a loss of gut microbes following ABX would attenuate exercise capacity. We further hypothesized exercise training to improve markers of mitochondrial function and biogenesis which will decrease following ABX.

METHODS

Study Design

This study was approved by the Institutional Animal Care and Use Committee (IACUC) of Rutgers, The State University of New Jersey. All experiments were performed in male 3–6-month mice (C57BL/6J from Jackson Labs; Farmington, CT but bred at Rutgers Medical School for several years) in a closed barrier vivarium. Each group of mice were assessed for maximal running distance using a graded exercise test on a mouse treadmill (Accuscan Instruments Inc. AN5817474) (14, 15) and tested PRE, POST, and after ABX treatment (Fig. 1). All animals were placed on standard chow (PicoLab Rodent Diet 20), which was irradiated to remove virtually all bacteria. All animals had ad libitum access to food and water throughout the study. Animals used in this study were maintained and all experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, Eight Edition, 2011) and all humane endpoints were followed in accordance with Rutgers IACUC.

FIGURE 1 –

Study design. Sedentary (PRE) C57BL/6J male mice were exercise trained for 60 min/d, 5d/wk for 5-weeks via forced treadmill running set at a constant 10° incline (POST). Graded exercise tests (GXTs) were performed on the same treadmill PRE, POST and after one-week of ad libitum access to antibiotic treated water (ABX). Mitochondrial biogenesis, oxidative phosphorylation and blood flow were evaluated at each time point.

Graded Exercise Testing

All mice were subjected to a practice trial 3 days before the experiments to adapt to the treadmill testing environment. At the time of the experiment, mice were placed on a treadmill with a 10% grade, which remained consistent throughout the test. The treadmill began at 4 m/min and the speed incrementally increased 2 m/min every 2 min until the mice reached exhaustion, which was defined as spending 10 seconds (10s) on the electric stimulus platform without attempting to reengage the treadmill belt.

Exercise Training

Exercise training consisted of forced treadmill running on the same treadmill as the initial test until exhaustion was reached. For the first 2 weeks of exercise training, the running speed was gradually increased until the mice could attain the desired workload (70% of maximal speed, 1 hour/day, 5 days/week for an additional 3 weeks).

Antibiotic Treatment

The antibiotic cocktail used to eliminate the gut microbes consisted of 0.3mg/ml of ampicillin (Sigma-Aldrich A1593), neomycin (Sigma-Aldrich 1458009), metronidazole (Sigma-Aldrich, M1850000) and vancomycin (Sigma-Aldrich, M1850000) in the drinking water at an average rate of consumption of 3–5 ml/day for 1 week. ABX treated water was continuously available and was replaced every 2–3 days. This dosing is consistent with the literature (16). The animals continued to be exercise trained during the ABX week to prevent detraining.

Hindlimb Blood Flow Assessment

Hindlimb blood flow was measured and quantified using the Vevo 3100 ultrasound system (VisualSonics Inc.), as previously described (17). Briefly, animals were anesthetized using sodium pentobarbital (60mg/kg i.p.) where an external jugular vein catheter was inserted into each mouse to facilitate blood flow measurements. The animals’ body temperature, heart rate, and respiratory rate were monitored and maintained within normal physiological ranges. Micro Marker non targeted contrast agents (VS-11913) were injected into the external jugular vein catheter of each mouse, microbubble concentration = 1 × 10⁸/50 μl 50μl injection/mouse, using an infusion pump set to a standardized rate of 600 μl/min. For hindlimb blood flow assessment, the mouse was in prone position, and the transducer was placed longitudinally over the right hindlimb, such that visualization of the gastrocnemius and bifurcation of the femoral artery into the popliteal and saphenous arteries were observed. Following data acquisition, analysis of the perfused myocardium and hindlimb was completed by using the VevoCQ software (Visual Sonics Inc.). The region of interest encapsulated the entire gastrocnemius muscle for the hindlimb blood flow assessment.

Biochemical Assays

Mice were sacrificed within 2 hours following their final bout of exercise training. The quadriceps muscle was removed and snap frozen immediately and the tissue was stored at −80 C°. The tissue was then homogenized, and protein concentration was determined using a Pierce BSA protein assay kit (Thermo Fisher; 23225). Citrate synthase activity was then measured using a citrate synthase activity kit (Cayman Chemicals; 701040). Complex IV activity was measured using a Complex IV rodent assay kit (Cayman Chemicals; 700990). Total nitric oxide expression was measured using a Total Nitric Oxide Assay (Invitrogen; EMSNO). Sirtuin-1 (SIRT1) activity was assessed by a fluorometric assay (Abcam; ab156065). AMP Kinase phosphorylation was measured using an ELISA (LSbio; LS-F36058O). All assays were performed as recommended by the manufactures.

PGC-1α protein concentration was assessed with a Pierce BCA Protein Assay Kit (Thermo Scientific catalog: PI23227). Samples were loaded onto 10% Tris·HCl gels (Thermo Scientific) transferred to nitrocellulose membranes and stained with primary antibodies (abcam; ab54481) and reacted with a goat-anti rabbit secondary antibody (abcam; ab6721). Protein abundance was expressed as relative units normalized to β-actin (abcam; ab8227) and quantified with Image Digits Studio (version. 5.0).

Statistical analysis

All data are expressed as mean ± standard error (SEM). Statistical comparisons were calculated using a one-way ANOVA, with a Tukey’s post hoc test. P values of <0.05 were considered significant. All statistical analysis was performed in Prism 9 for Windows and MacOS and SPSS (V26).

RESULTS

Body and Organ Weights

Exercise training did not alter body weight however ABX significantly decreased body weight compared to PRE and POST (Fig. 2A, p<0.001). While exercise training did not alter cecal weight, ABX significantly increased cecal weight in mice PRE (p<0.001) and POST (p< 0.0001, (Fig. 2B)) and there was a significant reduction in the number of reads from PRE (300K reads) compared to POST ABX (13,490 reads) demonstrating that ABX was effective. These reads are for base pairs greater than 1000. Reads denotes the coverage on the basis of the number and the length of high-quality reads before or after alignment to the reference, coverage has also been used to denote the breadth of coverage of a target genome (18). As such, the lower the read number, the less breadth and quality of the targeted genome to sequence.

FIGURE 2 –

(A) ABX treatment significantly decreased body weight compared to –POST (p<0.001). (B) Exercise training did not alter, where ABX treatment significantly increased cecal weight compared to –PRE (p<0.001) and –POST (p<0.0001). ABX significantly decreased liver (2C, p<0.0001) and skeletal muscle (quadriceps, 2D, p<0.0001) weight compared to POST. Significantly reduced skeletal muscle weight was persistent even after correcting for cecal weight (2E, p<0.05)

Reductions to liver, and skeletal muscle weight with free access to ABX water were also seen (Fig. 2C, D). A significant increase was seen in liver weight following exercise training (Fig. 2C, p<0.01) however, mice showed significantly lower liver weight post ABX (Fig. 2C, p<0.0001). A significant reduction (p<0.0001) was seen in muscle with ABX reducing quadriceps weight compared to –PRE (Fig 2D, p<0.0001) and –POST (Fig. 2, p<0.0001). Even when corrected for cecum weight, since it is elevated in ABX, skeletal muscle weight remained significantly lower after ABX compared to POST (Fig. 2E, p<0.05).

Exercise capacity.

Exercise training improved maximal running distance in mice by 60 ± 13% (Fig. 3A, p<0.0001) and work to exhaustion 63 ± 11% (Fig. 3B, p<0.0001). Both increases in maximal running distance and work to exhaustion induced by exercise training were abolished by ad libitum access to ABX water (Fig. 3, p<0.001).

FIGURE 3 –

5 weeks of exercise training (POST) significantly increased maximal running distance and work to exhaustion in mice. Ad libitum access to ABX was used to eliminate the gut microbiota resulting in abolition of the increases in maximal running distance (A) and work to exhaustion (B) induced by exercise training. Values represent mean ± SEM. A one-way ANOVA with a Tukey post hoc test was used to show significance at *** p<0.001 and **** p<0.0001.

Hindlimb Blood Flow:

Consistent with exercise training adaptations, hindlimb blood flow increased after exercise training by 80 ± 18% (p<0.01, Fig. 4 A,B). ABX eliminated the increases in hindlimb blood flow induced by exercise training (p<0.05, Fig. 4 A,B).

FIGURE 4 –

Exercise training significantly increased hindlimb blood flow (gastrocnemius) in mice and ABX abolished these increases (A). Representative figures showing levels of blood flow measured ultrasonically in the mice hindlimb (B). A one-way ANOVA with a Tukey post hoc test was used to show significance at * p<0.05, ** p<0.01.

Mitochondrial Function.

AMPK was significantly increased, by 136 ± 21%, with exercise training (p<0.0001, Fig. 5A). SIRT 1, a protein, which can be upregulated by AMPK, and is involved in mitochondrial biogenesis and function, was increased by 58% following exercise training (p<0.05, Fig. 5B). Consistent with the increase in SIRT 1 we found that protein expression of skeletal muscle PGC-1α was increased by 96% following exercise training (p<0.001, Fig. 5C). ABX treatment eliminated the increases of AMPK, SIRT1 and PGC-1α induced by exercise training.

FIGURE 5 –

Markers of mitochondrial biogenesis (A) SIRT1, (B) AMPK, and (C) PGC-1α were increased (p<0.05) in mice following 5-weeks of exercise training. These increases were abolished following ABX administration. Values represent mean ± SEM. A one-way ANOVA with a Tukey post hoc was used to show significance at * p<0.05, *** p<0.001 and **** p<0.0001.

Citrate synthase activity was increased by 49 ± 10% following exercise training (p< 0.05, Fig. 6A). In another marker of mitochondrial function Complex IV activity was also increased with exercise training, by 124 ± 11% (p<.0001, Fig. 6B). Both increases in citrate synthase activity and Complex IV activity induced by exercise training were abolished by ABX.

FIGURE 6 –

Exercise training improved mitochondrial function and skeletal muscle nitric oxide activity. Markers of mitochondrial function (A) citrate synthase and (B) Complex IV activity were both increased in mice following exercise training. This effect was abolished following ABX administration. (C) Skeletal muscle nitric oxide activity increased following exercise training (p<0.05) and was also abolished following ABX. Values represent mean ± SEM. A one-way ANOVA with a Tukey post hoc was used to show significance at * p<0.05, *** p<0.001, **** p<0.0001.

Nitric Oxide activity is also linked to mitochondrial function. So, it was not surprising that nitric oxide activity increased by 72 ± 17% following exercise training (p<0.001, Fig. 6C). ABX abolished the increases in nitric oxide induced by exercise training.

DISCUSSION

Summary

The major findings of this investigation involve two mechanisms (Fig. 7) that mediate training-induced enhanced exercise performance (blood flow and mitochondrial function) and those mechanisms mediating loss of training-induced enhanced exercise capacity with the deletion of the gut microbiota using ad libitum access to antibiotic treated water. Specifically, we showed that exercise training increased exercise capacity as reflected by a 60% increase in running distance and a 63% increase in work to exhaustion, and that elimination of gut microbiota eliminated the increased exercise capacity, which is consistent with what others observed (19, 20). However, many of the mechanisms we found mediating these effects, e.g., hindlimb blood flow and mitochondrial function, are novel.

FIGURE 7 –

Mechanisms Mediating Enhanced Exercise Capacity Induced by Exercise Training. Exercise capacity is increased due to increased hindlimb blood flow mediated by increases nitric oxide and increased mitochondrial oxidative phosphorylation (citrate synthase, complex IV) providing the stimulus to increased mitochondrial biogenesis (AMPK, SIRT1, and PGC-1α). ABX in drinking water abolished the increases in the markers of mitochondrial oxidative function (citrate synthase, complex IV, and nitric oxide) and the increases in mitochondrial biogenesis (AMPK, SIRT1, and PCG-1α). Thus, the elimination of the gut microbiota, using ABX, eliminated the increased running distance and work to exhaustion induced by exercise training and the mechanisms mediating the improved exercise capacity induced by exercise training.

Body and Organ Weight and ABX

The current study found that ABX significantly reduced body, liver, and skeletal muscle (quadriceps) weights. Other studies have also reported decreases in body, liver, and quadriceps weight following ad libitum access to antibiotics (21–24). Hill et al., (21) demonstrated a 60% decline in body weights of mice after two weeks of ABX administration in drinking water, which was restored after two weeks of being placed back on regular drinking water. Wu (24) specifically noted that within 24 hours of ABX liver weight was significantly reduced, which persisted for the remainder of the 7 days when ABX was administered. Wu (24) further showed that ABX inhibited liver regeneration and function. Liver is critical during exercise for the mobilization and breakdown of glycogen for maintenance of blood glucose levels suggesting that a reduction in its weight may interfere with its ability to function, particularly during exercise (25). Finally, Nay et al., (22) showed that with 10 days of ABX both muscle (gastrocnemius and quadriceps) and liver weight was significantly reduced. Furthermore, they showed that when animals underwent natural reseeding, the restoration of the gut microbiota, all muscle and liver weights increased to baseline levels. Unlike the current study, when Nay et al., (22) normalized for cecum weight the decrease in muscle weight was longer significant. However, Valentino et al., (26) reported similar findings in that despite increased cecal weight with ABX, this did not influence skeletal muscle weight and noted reduced muscle weight with ABX. Current evidence supports a role of the gut microbiota in maintenance of body and organ weights, including liver and skeletal muscle, critical organs for endurance exercise.

Exercise Capacity and ABX

Studies have shown ABX administration can reduce exercise capacity in mice (22, 23) while others have not (26). Still, others have shown reductions in exercise capacity in females from 4 replicate high runner lines of mice, bred for voluntary exercise compared with 4 non-selected control lines (19). Nay et al., examined male mice (14 wk. old) who were randomly divided into either a control group, or antibiotics for 10 days, followed by 10 days of natural reseeding of gut communities (22). While they reported no change in maximal aerobic velocity, they did find endurance limit time to be significantly reduced in ABX treated mice. Furthermore, Nay reported increased fatigability of extensor digitorum longus using ex vivo contractile tests. Both endurance limit time and fatigability were restored with natural reseeding, suggesting a role for gut microbiota in skeletal muscle endurance and fatigue. Valentino et al., (26) and McNamara et al., (19) showed no influence of ABX on exercise capacity in their female mice. However, Valentino et al. (26) reported inhibition of muscle hypertrophy after ABX, as their progressive weighted wheel running protocol, is specifically designed to investigate this phenomenon. Furthermore, while McNamara et al. (19) reported control female mice not to have a decline in performance with ABX those female mice bred to be high runners showed significant (21%) reduction in voluntary wheel running. A glaring difference between those studies and the current study is sex of the animal. Our study used male mice, while the others used female mice. These differences may allude to a sex difference in the contribution of the gut microbiota on exercise tolerance that must be investigated in future studies. The other major difference is that these studies provided ABX during the exercise training period, while this study did not, rather asking the question if ABX interferes with exercise capacity and two mechanisms which mediate enhanced exercise capacity highlighting the novelty of this study and its findings.

Hindlimb Blood Flow

One novel mechanism, not shown previously, is that hindlimb blood flow was significantly increased with exercise training and was no longer different from pre-exercise training levels after ABX. Given the extensive literature surrounding the salutary effects of chronic exercise in cardiovascular health and disease, including healthful aging, diabetes, obesity, atherosclerosis (27), and peripheral artery disease (28), examination of mechanisms modulating blood flow and its links to the gut microbiota are clinically relevant.

Mitochondria Markers of Oxidative Phosphorylation and Biogenesis

The endosymbiotic theory of mitochondrial origin is well confirmed, taking place about 1.5 billion years ago and related to the increase of O2 level in the atmosphere (29). The theory states mitochondria are ancestors of the ancient endosymbiotic organisms (the host) and the symbiont resembling bacteria as we know them today. The mitochondria have evolved to be the organelle we study today, but due to its bacterial-derived origin, it is no surprise that there is a gut-mitochondria axis that requires attention. Mitochondria play a crucial role in energy production through the synthesis of ATP via oxidative phosphorylation and it is well known that exercise training improves mitochondrial function through the PGC-1α and AMPK-SIRT1 pathway, regulating energy metabolism and mitochondrial biogenesis (14, 30), and is consistent with our data (Figs. 5 and 6). However, our data demonstrate that the improvement in mitochondrial function are abolished by ABX, which is novel. We found that mitochondrial biogenesis improved with exercise training, as reflected by a 136% increase in AMPK protein expression, a 58% increase in SIRT1 activity in quadriceps muscle lysate and a 96% increase in PGC-1α protein expression. These increases in mitochondrial function may result in increased glucose and fatty acid uptake and oxidation in skeletal muscle (26). Furthermore, as exercise training increased mitochondrial biogenesis (and thus mitochondrial function), our observation that ABX eliminated the adaptation in mitochondrial functional proteins also explains the reduction in exercise capacity. In further support of our findings that mitochondrial function is improved in exercise through the gut microbiota, it has also been suggested that changes in mitochondrial gene regulation and function may be mediated through microbiota-linked signal transduction (31). Butyrate, a gut microbial metabolite, is a key regulator of energy production and mitochondrial function by inducing PGC-1α gene expression in skeletal muscles and brown adipose tissue (32) and improving respiratory capacity and fatty acid oxidation via the AMPK-ACC pathway activation (33). Furthermore, in skeletal muscle cells, butyrate phosphorylates AMPK and p38 which then activates PGC-1α and thus fatty acid oxidation and ATP production.

AMPK is increased by exercise training (34) and may mediate enhanced exercise capacity. Additionally, as noted above, gut microbial metabolites have been shown to interfere with the AMPK pathway and thus the potential activation of PGC-1α. A disruption in PGC-1α activation would reduce mitochondrial biogenesis and contribute to an overall reduction in exercise capacity following ABX. This is also the first study that shows the exercise training induced increases in skeletal muscle AMPK to be disrupted with ABX, formally linking the gut microbiota to this enzyme.

PGC-1α has been reported to be the most dominant regulator of mitochondrial function and respiration in muscles (35), especially during endurance exercise (36–38). Additionally, PGC-1α is involved in thermogenesis, glucose metabolism and oxidative capacity in various tissues and can be phosphorylated by AMPK, also involved in mitochondrial biogenesis. Given the important interrelationship among PGC-1α, AMPK, and SIRT1 as well as the link to gut microbial metabolites influencing this pathway, it was important to not only examine PGC-1α, but also AMPK and SIRT1.

SIRT1, a redox sensitive energy sensor, can also affect mitochondrial biogenesis via PGC-1α deacetylation (39, 40). It is known that increased expression, content, and activity of sirtuins (41, 42). We found a 58% increase in skeletal muscle SIRT1 activity following chronic exercise training, which was abolished by ABX.

Citrate synthase activity and complex IV are mitochondrial enzymes within the electron transport chain, which is the major site for energy (ATP) production within the cell. Citrate synthase activity is routinely used as a marker of aerobic capacity and mitochondrial density in skeletal muscle (43, 44). The current study showed that citrate synthase activity increased by 49%, as expected with exercise training (14). However, the increase in citrate synthase activity was abolished by ABX. Complex IV has also been closely associated with mitochondrial oxidative phosphorylation capacity (43). Larsen et al. reported that complex IV activity was the biomarker that had the highest concordance with muscle oxidative phosphorylation due to its consistent correlation with many of the respiratory measures in diverse protocols (43). The current study, consistent with the literature (14), showed that complex IV activity increased with exercise training. However, our results showing that the increases in complex IV with exercise training were abolished following ABX has not been shown before. Given this marker has been reported to the best reflection of mitochondrial oxidative phosphorylation, the reduction seen with ABX to eliminate the gut microbiota, supports the concept that the gut microbiota may be required for exercise induced adaptations to mitochondrial oxidative function.

Finally, we examined skeletal muscle nitric oxide activity following chronic exercise training. The most important function of nitric oxide is its role in vasodilatation, blood flow, and mitochondrial respiration, resulting in enhanced exercise performance (45, 46). We found that exercise training increased skeletal muscle nitric oxide activity, by 72%, which was abolished following ABX. Given the role of nitric oxide in both mitochondrial function and regulation of blood flow, it is plausible to reason that the reductions not only seen in mitochondrial function, but also blood flow may be related to the decrease in nitric oxide activity with ABX. As with the other biomarkers, it is reasonable to hypothesize that gut microbial metabolites may be involved given the role in those metabolites in cardiovascular function, as described above.

Limitations

The current study did not include a female cohort due to animal availability at the time of this study however, as noted above future studies should aim to include female mice to examine if the impact of ABX on exercise capacity is sex specific as well as employ metabolomics to examine the impacts of exercise, gut microbiota, and its depletion on the metabolome. Furthermore, the current study did not include a group of animals on ABX that were not exercise trained, follow up studies aimed to examine the effects of ABX on muscle function independent of exercise would be beneficial to fully elucidate the role of the gut microbiota in muscle.

Practical Application

A difference between the current body of literature and our study is that ABX was given during exercise training, while our study gave ABX after training to understand if ABX impacts established adaptations to exercise, which is unique and novel. Given the novelty of this study and its findings, there should be important research designs aimed specifically to examine how ABX can impact an athlete’s performance since they are so often prescribed antibiotics (47). Ferry et al., (2020) noted in their review that studies have shown that 44% of track and field athletes of various ages and nationalities reported use of at least 1 antibiotic and from 2002 to 2014, nearly 70% of the participants in the men’s FIFA World Cup used an antibiotic at some point during the tournament (48, 49). Future studies examining the safety and effectiveness of drugs, including antibiotics, in athletes of all levels is needed, particularly in the gut microbiota to better educate clinicians and athletes on optimal use, potential side effects, and countermeasures to combat potential decrements in performance.

CONCLUSIONS

The current investigation demonstrated that the gut microbiota plays an important role in mediating the increases in exercise capacity after exercise training, which, in turn, is mediated by increases in aerobic capacity, increases in hindlimb blood flow and mitochondrial function. ABX eliminated the increased exercise capacity following exercise training and reversed the augmented mitochondrial biogenesis and blood flow which could be mediating the increases exercise capacity. Our data support the concept that ABX reduces the gut microbiota and decreases oxidative phosphorylation and counters mitochondrial biogenesis and thus limits exercise capacity. Thus, an intact gut microbiota is crucial for mediating exercise-induced training adaptations for the host, impacting exercise capacity, hindlimb blood flow, and mitochondrial function. This study highlights the practical need to understand how ABX usage among humans (athletes) may impact aerobic exercise training and further what could be done to offset the impacts of ABX usage.