Effects of oral HPΒCD-angiotensin-(1-7) supplementation on recreational mountain bike athletes: a crossover study

ABSTRACT Background Supplementation with Angiotensin-(1-7) [(Ang-1-7)] has received considerable attention due to its possible ergogenic effects on physical performance. The effects of a single dose of Ang-(1-7) on the performance of mountain bike (MTB) athletes during progressive load tests performed until the onset of voluntary fatigue have previously been demonstrated. This study tested the effects of Ang-(1-7) in two different exercise protocols with different metabolic demands: aerobic (time trial) and anaerobic (repeated sprint). Methods Twenty one male recreational athletes were given capsules containing an oral formulation of HPβCD-Ang-(1-7) (0.8 mg) and HPβCD-placebo (only HPβCD) over a 7-day interval; a double-blind randomized crossover design was used. Physical performance was examined using two protocols: a 20-km cycling time trial or 4 × 30-s repeated all-out sprints on a leg cycle ergometer. Data were collected before and after physical tests to assess fatigue parameters, and included lactate levels, and muscle activation during the sprint protocol as evaluated by electromyography (EMG); cardiovascular parameters: diastolic and systolic blood pressure and heart rate; and performance parameters, time to complete (time trial), maximum power and mean power (repeated sprint). Results Supplementation with an oral formulation of HPβCD-Ang-(1-7) reduced basal plasma lactate levels and promoted the maintenance of plasma glucose levels after repeated sprints. Supplementation with HPβCD-Ang-(1-7) also increased baseline plasma nitrite levels and reduced resting diastolic blood pressure in a time trial protocol. HPβCD-Ang-(1-7) had no effect on the time trial or repeat sprint performance, or on the EMG recordings of the vastus lateralis and vastus medialis. Conclusions Supplementation with HPβCD-Ang-(1-7) did not improve physical performance in time trial or in repeated sprints; however, it promoted the maintenance of plasma glucose and lactate levels after the sprint protocol and at rest, respectively. In addition, HPβCD-Ang-(1-7) also increased resting plasma nitrite levels and reduced diastolic blood pressure in the time trial protocol. Trial registration RBR-2nbmpbc, registered January 6th, 2023. The study was prospectively registered.

Recently, RAS was also found to play a key role in local perfusion and metabolism in skeletal muscles [4].Motta-Santos et al. [5] showed that ACE2 was responsible for the production of Ang-(1-7) from Ang II, ACE2 deficiency affected physical performance and cardiac and skeletal muscle induced by voluntary running training.
In the skeletal muscle, Ang-(1-7) activates the PI3K/AKT pathway in endothelial cells, increases glucose uptake, and improves insulin resistance through its antioxidative stress effect [6].With regard to exercise, recently, the Mas agonist A779 was shown to abolish the insulin sensitizing effect of exercise in skeletal muscle, suggesting that Ang-(1-7) participates in exercise-induced enhancement of insulin sensitivity [7].Recent research in our laboratory has also demonstrated the ergogenic effects of the oral formulation of HPβCD-Ang- (1)(2)(3)(4)(5)(6)(7).Oral ingestion of this formulation has been associated with a reduction in acute pain perception and improved maximum strength levels in young individuals trained following eccentric exercise-induced muscle injury [8].In another study, recreational performance of mountain bike (MTB) athletes supplemented with the HPβCD-Ang-(1-7) formulation showed reduced subjective perceived exertion, as well as increased total exercise time, oxygen consumption, and mechanical efficiency in a progressive test performed until voluntary fatigue [9].Furthermore, compared to nontransgenic animals, transgenic animals with higher circulating levels of Ang-(1-7) 2.5 showed fewer reductions in plasma glucose levels and depletion of muscle glycogen levels after exhaustive exercise, suggesting a decrease in muscle and liver glycogen storage [10].
Physical performance associated with supplementation in cyclists has been widely investigated [11][12][13].Taking into account the characteristics of MTB, the sprint mode is demanding on physical performance, and the aim of supplementation with HPβCD-Ang-(1-7) is to improve physical performance [14].The aim of this investigation was to evaluate the effects of an Ang-(1-7) formulation on the physical performance of recreational MTB athletes using two exercise protocols: the time trial and repeat sprint, by evaluating performance, fatigue, cardiovascular parameters and recovery.

Methods
This study was approved by the Ethics Committee on Human Research of the Federal University of Ouro Preto (protocol number:14912519.4.0000.5150).All procedures followed the Brazilian guidelines and standards for human research.Participants were informed about the research objectives, the steps to be taken, and the risks and benefits of their participation.Those who agreed to participate signed an informed consent form.

Volunteers
Twenty-one recreational MTB athletes, from the Ouro Preto-MG region, were selected.Participation was voluntary and the inclusion criteria were established as follows: male athletes over 18 years old, who had practiced MTB for at least 3 years, with a weekly training volume of at least 5 days a week, and participation in regional/national races over the last 6 months.The exclusion criteria were nonattendance to the battery of physical tests on the scheduled day and time; any illness that could compromise data collection; individuals with any type of pathology, smokers, use of medications or supplements, such as anti-inflammatory drugs and antibiotics, and/or other treatments that could compromise research data.

Study design
Participants were evaluated on three separate occasions (M1, M2, and M3), with a weekly interval between sessions.At M1, volunteers signed the Informed Consent Form and were familiarized with the experimental protocols.Subsequently, 10 participants were assigned to the time trial group, whereas the other 11 participants were assigned to the repetitions group.The participants then completed the M2 and M3 phases, which consisted of running a time trial or repeating sprints after ingesting placebo or HPβCD-Ang-(1-7).A 7-day washout period was applied between placebo and HPβCD-Ang-(1-7) (crossover).Half of the participants randomly used HPβCD-Placebo in M2 and HPβCD-Ang-(1-7) in M3, and vice versa.The randomization of the physical test protocols was conducted by a researcher who was not involved in data collection.An anamnesis was collected to determine if there were any adverse reactions or maintenance of diet and physical training routines.Immediately after the testing protocols, blood and urine samples were collected and the heart rate (HR) and blood pressure (BP) were measured at rest and during peak exercise.Figure 1 shows the study flow chart.

Supplementation
Supplementation was double-blinded and randomized.Participants received the HPβCD-Ang-(1-7) (0.8 mg) or HPβCD-Placebo (0.8 mg) formulations, which were administered orally.Both formulations were capsules (2 mg) and were manufactured at the Laboratory of Hypertension (Institute of Biological Sciences of the Federal University of Minas Gerais), three hours before the beginning of each test protocol.It should be noted that our research group patented the use of the formulation [(HPβCD-Ang-(1-7)] under number (BR 10 2016 0244064).The ingestion time (i.e. 3 hours before exercise) was established considering the window of action of Ang-(1-7) that is between 2 and 6 hours [15].The capsules were identical in appearance, size, weight, and taste, ensuring blinding of the participants.Double-blinding and randomization of the experiments were performed by a researcher who was not involved in data collection.The groups were revealed to the other researchers only at the time of data interpretation.
During the intervention, regular physical training, food intake, and duration of sleep were required.Additionally, individuals were instructed not to ingest any food and/or supplements that could alter cardiovascular parameters (such as caffeine and guarana) and to avoid strenuous exercise in the 48 h preceding the tests.

Anthropometric evaluation
Anthropometric evaluation was performed using a digital scale with a precision of 100 g and a maximum capacity of 150 kg (G.TECH®).A stadiometer with a 0.1 cm scale (WISO®) was used to measure the height.These measures were used to calculate the body mass index (BMI) using the equation

Blood pressure and heart rate assessment
BP was measured using an aneroid sphygmomanometer (Missouri®) with a stethoscope (Missouri®), before and immediately after the testing protocols.The HR at rest and the maximum exertion was assessed using the Polar RS800 (POLAR, Finland).

Acquisition of electromyographic signals
During repeated sprints, electromyographic signals (EMG) activity of the right vastus lateralis and medialis muscles was recorded at a sampling rate of 2000 Hz using a 24-bit A/D converter (EMG System, São Jose dos Campos, Brazil).Self-adhesive Ag/AgCl surface electrode pairs (model 2223BRQ, 10 mm diameter 3 M®), with an interelectrode distance (center-to-center) of 20 mm, were placed in the belly of the vastus lateralis and medialis muscles according to the Surface Electromyography for the noninvasive assessment of muscles standards [16].The reference electrode was positioned in a neutral location (tibia).To minimize any unwanted movements and any interference, we fixed the electrodes on the skin using an adhesive tape.The skin was shaved and cleaned with isopropyl alcohol to reduce impedance.The electrode positions were marked with indelible ink to ensure identical placement in all experimental trials.
The EMG signal was amplified with an octal bioamplifier (input impedance = 1 GΩ, common mode rejection ratio = 100 dB, gain = 2000) and analyzed offline using a custom-made MATLAB software program.The raw EMG signal was filtered using secondorder Butterworth bandpass filters (cutoff frequencies set at 20 and 500 Hz) to remove noise and artifacts.The root mean square (RMS) was calculated for every burst during sprints, averaged at 5s epochs, and normalized by RMS at the first 5-s epoch.

Determination of the maximal VO 2
Participants in the time trial group had a fourth meeting (M4) to determine their maximum oxygen consumption (VO 2 max).A progressive test was performed using a leg cycloergometer (Biotec 2100, CEFISE Biotechnology) in an open circuit using VO2000® equipment (VO2000, MedGraphics®, Saint Paul, Minnesota, USA).Volunteers started with a 5-minute warmup with a load of 100 W. The workload increased by 25 W per minute until voluntary fatigue or cadence decreased below 50 rpm [17].The VO 2 max value was determined by averaging values of the last 2 minutes [9].

Physical test protocols
Time trial (TT20km): A cycle ergometer (Biotec 2100, CEFISE Biotechnology) was connected to a device (Ergometric 6.0, CEFISE Biotechnology) that provided power (W), cadence (rpm) and speed (km.h −1 ).The cycle ergometer was adjusted individually according to the preferences of the volunteers.The device was calibrated prior to each test according to the manufacturer's instructions.After a 7-minute warm-up at a fixed load of 1 kg (free rpm), participants immediately started the TT20km cycling at a fixed load of 1.5 kg throughout the trial [18,19].Cyclists were instructed to complete the test in the shortest possible time.During the test, cyclists were allowed to rise from their seats and were free to track the rpm and km/h.The time taken to complete the TT20 km, and the average power.The RPM and the speed were recorded and used as performance measures.A 500 mL bottle of water was provided during the hydration tests to avoid differences in consumption among the participants.
Repeated sprint: Individuals performed a 10-minute warmup, with free rotation and 1 kg resistance on a cycle ergometer for legs (Biotec 2100, CEFISE Biotechnology).After warm-up, the load was set individually for each volunteer (7.5% of the body mass) [20,21], and the participants performed four 30-s sprints with a passive pause of 5 min between sprints [22].The mean power (W mean), maximum power (Wmax), and fatigue index were obtained using the Ergometric 6.0 software (CEFISE Biotechnology) [20,23].

Plasma and urinary analysis
Venous blood samples were collected before and immediately after exercising.Vacutainer tubes containing sodium fluoride, empty tubes, or EDTA were used.The concentration of lactate, glycemia, and the level of nonesterified fatty acids (NFAs) in plasma was evaluated using commercial kits (BIOCLIN FAB.QUIBASA QUÍMICA BÁSICA LTDA PROC.BRASIL) using an automatic analyzer (CM200, Wiener Lab Group, Rosario, Argentina).Midstream urine samples were collected at the beginning of each trial and immediately thereafter.Nitrite was measured in urine and plasma, and testing was performed by adapting the protocol [24], using a microplate reader in a 548 nm spectrum (BIOTEK, Synergy HT).

Statistical analysis
Statistical analyses were performed using GraphPad Prism 7 software (GraphPad Software Inc., San Diego, CA, USA).Data was described as mean ± standard deviation.Data normality was verified using the Shapiro-Wilk test.Inter-and intra-condition differences were compared using a paired t-test when the data had a normal distribution.For nonnormally distributed data, the Wilcoxon matched pair signed rank test was used.Information regarding the normality of the data was added to the figures.Data were expressed as mean ± standard deviation (SD) for data having a normal distribution, and nonparametric data were presented as median±95% confidence interval (CI).The significance level was set at p < 0.05 for all tests.The practical importance of pairwise comparisons was assessed by calculating Cohen's effect size d [25].Effect sizes (d) greater than 0.8, between 0.8 and 0.5, between 0.5 and 0.2, and less than 0.2 were considered large, moderate, small, and trivial, respectively [26].In addition, an ANOVA test (mixed effects) test was conducted for EMG analyses.

Results
Table 1 presents the characteristics of the participants.The data represent the mean and standard deviation values of the 21 male volunteers.All participants completed the planned experimental design.
To ensure the level of physical conditioning of the sample, the VO 2peak test was performed for individuals who underwent the time test (61.12 ± 12.78 mL.kg −1 .min−1 ) and a single Wingate peak and the mean power was obtained for those who underwent the repeated sprint protocol (7.2 ± 0.6 peak power W; 5.6 ± 0.6 mean power W).
As observed in Table 2, supplementation did not promote changes in cardiovascular parameters of individuals undergoing the repeated sprint protocol; however, participants who participated in the time trial protocol showed a reduction in diastolic BP values at rest (p = 0.025).
Regarding performance, there were no changes in values for the group submitted to the time trial (HPβCD -placebo 37.37 ± 3.2 min; HPβCD-Ang-(1-7) 37.04 ± 3.2 min; p = 0.28) (Table 3).Similarly, the group subjected to repeated sprints did not exhibit any statistically significant differences in peak power, mean power, or in the fatigue index (Table 4).
Regarding the biochemical parameters of the energy metabolites (Figure 3), there was an alteration in the plasma concentration values of lactate at rest (HPβCD-placebo 1.92 ± 0.2 mmol/L; HPβCD-Ang-(1-7) 1.68 ± 0.1 mmol/L; p = 0.014) for individuals who participated in repeated sprints (Figure 3b), but the same was not observed in individuals participating in the time trial (Figure 3a).Regarding plasma glucose values, the group that submitted repeated sprints (Figure 3d) under the supplement intervention did not show any changes after the exercise protocol, while in the placebo group, the changes were verified after the physical test (HPβCD-placebo rest: 87.7 ± 11.5 mg/dL, posttest:117.5 ± 25.9 mg/dL [p = 0.007]; HPβCD-Ang-(1-7) rest:93.9 ± 7.9 mg/dL, posttest: 99.4 ± 17.9 mg/dL [p = 0.073]).Conversely, the group subjected to the time trial did not show differences between conditions (Figure 3c).Regarding levels of non-esterified fatty acids (NEFA), none of the protocols produced significant differences compared to placebo or supplements, both at rest and after the physical test (Figures 3e,f).Finally, urinary nitrite levels in the group undergoing the time trial (Figure 3g) were shown to be higher at rest for the supplemented condition (HPβCD-placebo 9.25 ± 0.97 nmol; HPβCD-Ang-(1-7) 12.24 ± 3.60 nmol; p = 0.0402) and did not increase after the protocol (HPBCD-placebo 14.5 ± 5.54 nmol [p = 0.039]; HPβCD-Ang-(1-7) 18.1 ± 5.96 nmol [p = 0.4961]).Furthermore, no differences were observed between the exercise protocols with regard to plasma nitrite levels at rest or following the maximum effort between conditions (Figure 3i,j).

Discussion
Studies with evaluating HPβCD-Ang-(1-7) in humans using oral formulations are pioneering work and our group was the first to evaluate its effects on physical performance.This study exposed participants to 0.8 mg of the oral formulation of HPβCD-Ang-(1-7) as treatment.The aim of this study was to evaluate the effects of this formulation on physical performance, fatigue, recovery, and cardiovascular parameters.We hypothesized that the oral formulation could improve all the listed parameters.However, our results showed that for the repeated sprint group, cardiovascular parameters were not altered by the formulation; although the metabolic results showed a reduction in basal plasma lactate levels and the maintenance of plasma glucose levels, with no changes in NEFAs or nitric oxide (NO) levels before and after the repeated sprint protocol.Electromyographic recordings did not show any significant differences between conditions.Furthermore, the dosage did not affect the physical performance parameters evaluated.For the time trial protocol, the cardiovascular results indicated a significant difference in DBP at rest; although the same was not observed for the systolic BP or HR at rest or at maximum exertion.In addition, there were no changes observed in the plasma levels of NEFAs, lactate, or glucose.However, the baseline plasma nitrite levels increased.The physical performance parameters did not show significant differences; however, there was a 30-second reduction in test times for the supplemented group, which is a formidable result at the level of international MTB competitions, which are decided by a matter of seconds.
The HPβCD-Ang-(1-7) formulation significantly reduced resting DBP in the Ang-(1-7)-treated group in the time-trial protocol.These effects may be related to the vasodilator profile of Ang-(1-7) [27], and this vasodilation may have been promoted by a significant increase in basal NO levels in the group treated with the HPβCD-Ang-(1-7) formulation compared with the placebo condition.
The ACE 2/Ang-(1-7)/Mas axis induces the release of NO by increasing the enzymatic activity of endothelial NO synthase (eNOS).In skeletal muscle, Ang-(1-7) activates the PI3K/AKT pathway in endothelial cells, increases glucose uptake, and improves insulin resistance through its antioxidative stress effects [6].Recently, it was reported that the insulin sensitizing effect of exercise was abolished by the Mas agonist A-779 in skeletal muscle, suggesting that Ang-(1-7) participates in exercise-induced enhancement of insulin sensitivity [7].
In addition to NO release, Ang-(1-7) potentiates the activity of bradykinin (BK), another potent vasodilator [28].BK participates in physical performance by interacting with NO.These data strongly suggest that Ang-(1-7) can enhance the muscle microvasculature and increase microvascular endothelial surface area, leading to increased nutrient delivery to the skeletal muscle [29].Based on the hypothesis that Ang-(1-7) affects muscle microvasculature recruitment, we investigated the pattern of electromyographic activity in the vastus lateralis and medialis muscles after repeated sprint protocols and found no differences in responses in the supplemented condition.
In addition to regulating NO production in muscle, the AKT pathway participates in glucose transport through GLUT-4 translocation, protein synthesis through activation of translational components, lipogenesis through activation of transcription enzymes that catalyze the formation of long-chain fatty acids, and increases glycogen synthesis by inactivation via phosphorylation of glycogen synthase kinase 3 (GSK) [30].Therefore, when GSK-3 is phosphorylated by AKT, pyruvate dehydrogenase is active and ATP synthesis is directed to the citric acid pathway [31].
Altogether, previous findings have led to the generation of a physiological hypothesis whereby Ang-(1-7) administration could increase the availability and/or oxidation of free fatty acids.Consequently, this would result in improved overall metabolic efficiency with higher physical performance, especially in the trial protocol.However, no differences were observed between the HPβCD-Ang-(1-7) and HPβCD-placebo conditions in either exercise protocol in terms of plasma levels of NEFAs.
However, volunteers who integrated the protocol did not any show reductions in absolute average power production in the intervention supplemented with the HPβCD-Ang-(1-7) formulation, whereas in the placebo condition, reductions were observed.Supplementation may have helped in the recovery processes between exercise sessions, as oxidative processes are determined by ATP production to replenish creatine phosphorus stores [32].During the Wingate test, 70-80% of ATP production comes from anaerobic pathways [33][34][35][36].
Regarding changes in glucose and lactate levels, vasodilator activity produced by Ang-(1-7) through the Mas receptor may have played a key role in metabolic control, especially in glucose control, as transgenic mice with high levels of circulating Ang-(1-7) exhibited better insulin tolerance and improved insulin-stimulated glucose uptake [37].In the muscle, infusion with Ang-(1-7) increases glucose uptake via the GLUT-4 receptor [29].Furthermore, recent data from our group showed that transgenic mice, which have higher levels of circulating Ang-(1-7) (2.5-fold higher), when subjected to intense exercise, exhibited lower plasma glucose variations and less depletion of liver and muscle glycogen [8].Recently, it has also been shown that in healthy rats, an exercise session improves vascular insulin sensitivity on exposure to Ang-(1-7) [38].The data from this study support the above-mentioned findings, since after an anaerobic training session in plasma glucose levels observed following supplementation with the HPβCD-Ang-(1-7).Instead, supplementation with HPβCD-Ang-(1-7) was able to decrease the level of plasma lactate at rest, which is a by-product of anaerobic glucose metabolism [39].Further studies are needed to evaluate the relationship between Ang-(1-7) and lactate production.
Contrary to our study hypothesis, no changes in physical performance variables were observed in either the time trial or in the repeated sprint; however, supplementation with HPβCD-Ang-(1-7) was able to promote a reduction, albeit not significant, of approximately 30s, which is relevant in world MTB competitions whose outcomes are often decided by differences in seconds.Studies conducted in cyclists testing new supplements that increase performance found similar results, and the respective authors did not observe significant differences, but noticed an improvement in performance variables [12,40].It is important to note that a previous study in our laboratory showed a significant increase in total exercise time, oxygen consumption, and mechanical efficiency in a progressive test of voluntary fatigue with HPβCD-Ang-(1-7) supplementation in 14 athletes with MTB.However, in that study, the dose was twice as high as that used in the experiment [9].
There are some uncertainties about the optimal doseresponse relationship, as 0.8 mg supplementation with the formulation may not have been sufficient to alter plasma levels of Ang-(1-7) and detect improvements during the exercise interventions.Finally, although this study did not reveal that supplementation with the HPβCD-Ang-(1-7) formulation could improve physical performance in the two different protocols, new methodologies and dosages should be tested in other sports and athletes.Exposure to Ang-(1-7) may enhance the efficiency of nutritional input on performance and metabolism, which are of appreciated value in high-performance sports.Previous data [5] and evidence [8] suggest that this peptide exerts some effects on physical performance, but more studies are required to investigate the mechanisms involved.

Conclusions
Supplementation with HPβCD-Ang-(1-7) did not alter physical performance in the time trial or in the repeated sprint protocols of cyclists.However, supplementation was sufficient to avoid peaks in plasma glucose levels after the time-test exercise protocol and to reduce plasma lactate levels at rest in the repeated sprint protocol.Furthermore, a reduction in the diastolic BP and an increase in resting plasma nitrite levels were observed in the time trial group.Overall, these results indicate that the oral formulation HPβCD-Ang-(1-7) may influence physical performance and maintain glucose levels constant during exercise.The practical application entails the use of HPβCD-Ang-(1-7) as a performance-enhancing supplement with no side effects, especially in the cardiovascular system.

Table 1 .
Characterization of the sample.

Table 2 .
Cardiovascular parameters during rest and in maximal effort in time trial and repeated sprints protocols.

Table 3 .
Time trial performance variables.

Table 4 .
Repeated sprints results per stimulus.