Exploratory Study on Exercise Induced-Plasmatic Follistatin Kinetics : Proof of Concept

Background and objective: Follistatin is a protein that has been positively associated with increased myogenesis and decreased fat accumulation hence may be a potential target for preventing/treating obesity. Its expression is induced by exercise –which may, partly, explain the anti-obesity underlying metabolic pathways of physical activity-; however, not many studies have described its kinetics after performing validated exercise protocols that simultaneously evaluate the aerobic capacity. Thus we aimed to determine the plasmatic levels of exercise induced-follistatin and to correlate such with aerobic capacity. Materials and methods: After performing a treadmill stress test (Bruce protocol; with 3-minutes incremental velocity and inclination, until fatigue or reaching 85% maximal cardiac frequency), blood samples were drawn (at 1, 2, and 4 hours post-exercise) in order to asses follistatin kinetics. Results were correlated with maximum oxygen volume, calculated from protocol-validated equations. Results: 5 moderately active women volunteers were included (aged: 21 ± 0.7 years, weight: 54.7 ± 10.5 kg, VO2max: 57.4 ± 16.1 mL/ kg/ min). After stress test, a progressive decrease in the expression of follistatin during the first 2 hours was observed (m = -892); levels were found again increased at 4 hours. A negative correlation between aerobic capacity and exercise-induced follistatin levels were found. Conclusions: After exercise, follistatin levels exhibit a descending slope which, at 4 hours, again rises. The most relevant finding in this study was the negative correlation between aerobic capacity and follistatin expression, thus allowing the assumption that their metabolic and exercise-induced pathways may not be interrelated.


Background
Follistatin is an autocrine glycoprotein whose main role refers to the antagonism and inhibition of molecules belonging to the super family of Transforming Growth Factor Beta (TGF-β), whose clarified (although it has been suggested that the expression is rather hepatic vs. muscular-, which could be linked to the depletion of macronutrients that occurs during exercise) nor described the duration of such increase once the physical activity.
The objective of the present study was to therefore determine the plasmatic levels of exercise induced-follistatin during the recovery period and to correlate such with aerobic capacity.

Materials and methods
We conducted a proof of concept, including volunteer healthy young adults (20 -25 years old), with normal weight (body mass index > 18 y < 25 kg/ m 2 ), who performed moderately intense physical exercise regularly and signed the corresponding informed consent.Anthropometric assessment (weight and height) was performed according to Lohman's technique.Maximum Cardiac Frequency (MCF) and its correspondent 85% were calculated (MCF = 220 -age) in attention to the exercise protocol, as described in the following section.

Treadmill stress test
Bruce's protocol was used; in brief, this test progressively increases both velocity and inclination every 3 minutes (starting at 1.7 miles/ hour; 10% elevation) in 7 stages (reaching up to 6 miles/ hour; 22% elevation).The test finalizes whenever: a) total ergometry time (21 minutes) ends, b) subject refers "maximum fatigue" and "physical inability to continue the stress test" or, c) subject reaches his/ hers 85% of MCF.Whichever the case, total test time was recorded and further used to calculate aerobic capacity (i.e., VO 2 max) with the following formula: VO 2 max (mL / kg/min) = 4.38 × (time in minutes) -3.9 In addition, Heart Rate (HR) is also recorded immediately after finishing the stress protocol, 1 and 3 minutes afterwards.

Follistatin plasmatic levels
Before (i.e., prior to treadmill test), 1, 2, and 4 hours after completing stress test, venous blood samples were collected into plastic tubes containing citrate as anticoagulant.These were immediately centrifuged (3500 r.p.m., 15 minutes) and plasma was further separated into cryotubes.Follistatin determination was performed using an commercial immunocolorimetric kit (Abcam®).

Ethics
The present study complies with the ethical principles stipulated in Helsinki Declaration, the Nuremberg Code, and Mexican regulatory laws (Ley General de Salud en Materia de Investigación).In accordance to the latter, this research implies a "minimum risk" as procedures considered as "routinely" (weight measurement, stress test, blood collection < 450mL) are employed.Moreover, participants were informed of all procedures prior to any intervention and, finally, they could withdraw consent whenever they want to.

Statistical analysis
Quantitative variables are presented as mean ± standard deviation, unless otherwise stated; qualitative variables are presented as frequencies.For inferential analysis, repeated measures ANOVA with Bonferroni post hoc test were performed; correlations were evaluated according to Pearson.A statistical significance level of p < 0.05 was considered.All analysis was performed with GraphPad Prism®, v. 5.
After an open call, 5 subjects -all females, players of the soccer team-were recruited from a local academic institution (Universidad Anáhuac México, Campus Norte).Demographic and anthropometric data are show in Table 1.
Total test time was 14 ± 3.7 minutes, yielding an overall aerobic capacity of 57.4 ± 16.0 mL / min/kg.All but one of the participants reached their corresponding 85% of MHR (161.6 ± 21.0 bpm) thus stress test was ended.3 minutes post-test, finishing heart rate decreased, in average, almost 50% (83.0 ± 18.9 bpm) with a referred level of fatigue of 5 ± 1.7.
Plasmatic follistatin kinetics is shown in Figure 1.No significant differences were found among test times, although a decreasing slope (m = -892.0± 259.8, r 2 = 0.92) presented during the first two hours of recovery, whereas a perfectly fit increased gradient (m = 970.0± 948, r 2 = 1.0) was found in the late recovery (2-4 hours post-exercise).

Discussion And Concluding Remarks
Although not the initial aim of the present study, one of the main findings was that even though all volunteers were part of the University soccer team hence claimed to perform a moderatehigh level of physical activity (i.e., 2 hours of practice, 5 days a week), surprisingly, not one of the were able to conclude the stress test as they all reached 85% of MCF before completing 21 minutes of the Bruce protocol.But even more, standard deviations showed that results were greatly disperse among the recruited participants.This observation may be attributed to the fact that cardio-respiratory fitness and heart rate recovery have been associated with the in-field playing position of the study subjects [7], a confounding variable we did not take into account.
In this same thought, follistatin levels did not significantly differ from one time to another.This may be due to: a) small sample size and, b) wide variation that may be attributed to the previously mentioned basal cardio-respiratory fitness of each soccer player.Nevertheless, we found an ascending trend in follistatin plasmatic levels 4 hours after finishing the stress test.As ours, other studies have reported that the hormone's levels peak at 3 hours of recovery post acute exercise bouts [8].
Finally, even though a positive strong and significant correlation between VO 2 max and exercise was certainly expected, the negative correlation with the prior with basal follistatin was not.This is not widely reported within the literature, although a study reported no significant changes in blood follistatin after resistance exercise and, moreover, authors attribute such to the fact that levels of plasmatic myokines are enhanced by moderate intensity exercise, rather than by resistance (i.e., high intensity) training [9].This, in turn, may correlate with the types of muscle fibers (I or II): follistatin has been associated with type II (i.e., aerobic or slow twitching) fibers [10] thus may explain the aforementioned association with moderate (aerobic) exercise.

Table 1 :
Descriptive data BPM: Beats Per Minute; MHR: Maximum Heart Rate; SD: Standard Deviation