Experimental Physiology

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1 168 Exp Physiol (2016) pp Research Paper Research Paper Responses of sex steroid hormones to different intensities of exercise in endurance athletes Koji Sato 1,MotoyukiIemitsu 1, Keisho Katayama 2,KojiIshida 2, Yoji Kanao 3 and Mitsuru Saito 4 1 Faculty of Sport and Health Science, Ritsumeikan University, Kusatsu, Japan 2 Research Center of Health, Physical Fitness and Sports, Nagoya University, Nagoya, Japan 3 Faculty of Sport and Health Science, Tokaigakuen University, Nagoya, Japan 4 Faculty of Psychological and Physical Science, Aichigakuin University, Nisshin, Japan Experimental Physiology New Findings What is the central question of this study? What is the effect of different exercise intensities on sex steroid hormone concentrations in individuals with different degrees of physical fitness? What is the main finding and its importance? In endurance athletes, serum concentrations of sex steroid hormones increased only with high-intensity exercise. Moreover, different responses of sex steroid hormone secretions were induced by different exercise intensities in individuals with low and high levels of physical fitness. Previous studies have shown that acute exercise elevates sex steroid hormone concentrations in rodents and that sprint exercise increases circulating testosterone in healthy young men. However, the effect of different exercise intensities on sex steroid hormone responses at different levels of physical fitness is still unclear. In this study, we compared circulating sex steroid hormone responses at different exercise intensities in athletes and non-athletes. Eight male endurance athletes and 11 non-athletes performed two 15 min sessions of submaximal exercise at 40 and 70% peak oxygen uptake ( V O2 peak), respectively, and exercised at 90% V O2 peak until exhaustion. Venous blood samples were collected during the last minute of each submaximal exercise session and immediately after exhaustion. Acute exercise at 40, 70 and 90% V O2 peak induced significant increases in serum dehydroepiandrosterone (DHEA) and free testosterone concentrations in non-athletes. On the contrary, only 90% V O2 peak exercise led to an increase in serum DHEA and free testosterone concentrations in athletes. Serum 5α-dihydrotestosterone concentrations increased with 90% V O2 peak exercise in both athletes and non-athletes. Additionally, serum estradiol concentrations were significantly increased at moderate and high exercise intensities in both athletes and non-athletes. These results indicate that in endurance athletes, serum sex steroid hormone concentrations, especially serum DHEA and 5α-dihydrotestosterone concentrations, increased only with high-intensity exercise, suggesting that different responses of sex steroid hormone secretion are induced by different exercise intensities in individuals with low and high levels of physical fitness. In athletes, therefore, high-intensity exercise may be required to increase circulating sex steroid hormone concentrations. (Received 3 June 2015; accepted after revision 29 October 2015; first published online 31 October 2015) Corresponding author M. Iemitsu: Nojihigashi, Kusatsu, Shiga, Japan iemitsu@fc.ritsumei.ac.jp DOI: /EP085361

2 Exp Physiol (2016) pp Sex steroid hormone responses in athletes 169 Introduction Sex steroid hormones have important roles in improving muscle glucose metabolism (Sato et al. 2008), muscle protein synthesis (Ferrando et al. 2002) and antioxidant regulation (Jacob et al. 2010) and in influencing behavioural motivation and cognition (Pluchino et al. 2015) and affecting motor behaviour (Wegner et al. 2014). Testosterone and estradiol are mainly produced by the testes and ovaries as well as being converted from dehydroepiandrosterone (DHEA), which is secreted by the adrenal cortex. Testosterone is converted to 5α-dihydrotestosterone (DHT) in various tissues, such as the bones, heart, liver, brain and skeletal muscles (Labrie et al. 1995). 5α-Dihydrotestosterone has a high affinity for androgen receptors, and chronic elevation of DHT concentrations is related to metabolic and cardiovascular risk (Duskova et al. 2011). Exercise requires physical or mental exertion, especially when performed to develop or maintain fitness or as a means of practice or training. Investigation of sex steroid hormone concentrations at different intensities of exercise is important for the promotion of health and fitness and for determinating the effects of the intensity of exercise training. A few studies have reported acute exercise-induced alterations in serum sex steroid hormone concentrations. According to Kuusi et al. (1984), serum testosterone concentrations were decreased in physically active men after running on a treadmill using the Bruce protocol. On the contrary, repeated aerobic high-intensity exercise (consisting of 10 repetitions of 30 s sprinting at a target load of 150% of the maximal work capacity) increased serum total testosterone, free testosterone and DHT concentrations in healthy young men (Smith et al. 2013). Moreover, in highschool students after a session of high-intensity exercise (70 85% of maximal heart rate), testosterone (Budde et al. 2010) and cortisol concentrations (Budde et al. 2015) were significantly increased. The responses of sex steroid hormones to exercise might be influenced by the intensity of exercise. However, the effect of different intensities of exercise on DHEA, free testosterone, DHT and estradiol concentrations in individuals at different levels of fitness is still unclear. Understanding the changes in systemic sex steroid hormones during a single bout of exercise at different intensities may be helpful for understanding exercise training-induced adaptations. In athletes, endogenous anabolic hormones, such as DHEA, testosterone, DHT and estradiol, may play an important role in physiological adaptations to exercise training, i.e. muscle strength, mass and metabolism. Testosterone, DHT and estradiol are synthesized from DHEA through the activation of steroidogenic enzymes, such as 3β-hydroxysteroid dehydrogenase (HSD), 17β-HSD, 5α-reductase and P450 aromatase; therefore, the timing of blood sampling needs to be considered. Exercise-induced changes in sex steroid hormone concentrations tended to decrease within 1 3 h for the young healthy men in our preliminary study. Additionally, the hypothalamic pituitary adrenal axis controls adrenal androgens; therefore, measurement of exercise-induced changes in cortisol with different intensities of exercise would indicate the degree of adrenal activation. Therefore, the aim of the present study was to clarify the responses of sex steroid hormone and cortisol concentrations to low, moderate and high-intensity exercise of short duration in both athletes and non-athletes, measured immediately after exercise. To maintain and increase sex steroid hormone concentrations, enhancement of steroidogenesis by habitual exercise would be needed. Therefore, to estimate the capacity for steroidogenesis, we measured DHEA, free testosterone, DHT and estradiol concentrations immediately after each exercise session. We hypothesized that different intensities of short-duration aerobic exercise will induce different circulating sex steroid hormone responses in individuals with higher fitness levels compared with lower fitness levels. Methods Participants Eight male endurance runners [age 20.3 ± 0.3 years, peak oxygen uptake ( V O2 peak) 56.3 ± 0.8mlkg 1 min 1 ], who belonged to a collegiate track team, and 11 male non-athletes (age 22.0 ± 0.9 years, V O2 peak 42.1 ± 2.1 ml kg 1 min 1 ) participated in this study. All volunteers provided written informed consent before participating in the study, which was approved by the Ethics Committee of Nagoya University and conducted in accordance with the Declaration of Helsinki. The participants were examined by a physician to confirm that none had medical problems that might preclude participation or affect the results. Room temperature was maintained at C throughout the experiment. Measurement of V O2peak To determine participant fitness and to set the workload for subsequent submaximal exercise, all participants underwent an incremental exercise test to exhaustion using an electromechanically braked ergometer in a semirecumbent position (Aerobike 75XL III; Combi, Tokyo, Japan). The incremental exercise test began at an initial power output of 90 W, and the workload was increased 15 W every minute until exhaustion. The pedalling rate was maintained at 60 r.p.m. with the aid of a metronome. Minute expired ventilation, oxygen uptake

3 170 K. Sato and others Exp Physiol (2016) pp ( V O2 ) and carbon dioxide output were recorded during the testandwereaveragedevery30safterward.thehighest V O2 value obtained during the test was used as the peak V O2 ( V O2 peak). Respiratory variables were determined by an online system with a mixing chamber, as in our previous studies (Katayama et al. 2011, 2013). Thesubjects breathed through a leak-free nasal mask (5719; Hans Rudolph, Kansas City, MO, USA). Expired gas volume was measured using a Fleisch pneumotachometer (PN-230; Arco System, Chiba, Japan). Sample gas was drawn through a sampling tube connected to the pneumotachometer to measure expired gas fractions, which were analysed using a mass spectrometer (ARCO-1000; Arco System). Breath-by-breath data were analysed continuously using customized computer software (PC-9821Ra-40; NEC, Tokyo, Japan). Electrocardiography was recorded using a three-lead electrocardiograph (AB-621; Nihon Koden, Tokyo, Japan), and heart rate was calculated from each R R interval obtained from the electrocardiogram. Exercise protocol On a subsequent visit, both athletes and non-athlete participants completed two sessions of exercises on the ergometer, consisting of 15 min at 40% V O2 peak (low), 10 min of rest, 15 min at 70% V O2 peak (moderate), 10 min of rest and, finally, 90% V O2 peak until exhaustion (maximum). In order to normalize for circadian variations in the hormonal state, exercise took place in the morning between 2 and 4 h after waking. Blood sampling Fasting blood samples were obtained to avoid the effect of food consumption, as in our previous study (Sato et al. 2014). On arrival at the laboratory, venous blood from the antecubital vein was collected after 30 min of rest. Further samples were collected during the last minute of each submaximal exercise period and immediately after exhaustion. Serum and plasma samples were immediately centrifuged at 1500 g, 15min, 5 C. The supernatant was immediately transferred to polypropylene tubes and stored at 80 C until analysis. Sandwich enzyme immunoassay All techniques and materials used in these analyses were in accordance with the manufacturer s protocol. The serum concentrations of DHEA (Assay Designs, Ann Arbor, MI, USA), free testosterone (Cayman Chemical, Ann Arbor, MI, USA), DHT (IBL, Hamburg, Germany), estradiol (Cayman Chemical) and cortisol (Enzo Life Sciences Inc, Farmingdale, NY, USA) were determined using a sandwich enzyme immunoassay kit. The immobilized polyclonal antibodies were raised against DHEA, free testosterone, DHT, estradiol and cortisol, whereas the secondary HRP-coupled antibodies were monoclonal. Optical density at 450 nm was qualified using a microplate reader (xmark microplate spectrophotometer; Bio-Rad Laboratories, Hercules, CA, USA). All samples were assayed in duplicate, and all hormonal data were normalized by natural logarithmic transformation before analysis. Measurements were converted into concentrations using the linear fit of the log log plot of the standard curve. Statistical analysis All values are means ± SEM. Statistical analysis was performed using two-way repeated-measures ANOVA. A Bonferroni post hoc test was used to correct for multiple comparisons when the analyses revealed significant differences. For ANOVA, P < 0.05 was considered significant; P < 0.01 was considered significant for each post hoc test. Student s unpaired t test was used to compare characteristics between athletes and non-athletes. All tests were conducted with StatView version 5.0 (SAS Institute Inc., Tokyo, Japan). Results Physical characteristics Body weight, height and body mass index were not significantly different between athletes and non-athletes (Table 1). The V O2 peak was significantly higher in athletes compared with non-athletes (P < 0.01, t = 6.224), but no significant difference was seen in maximal heart rate (Table 1). Sex steroid hormonal responses Baseline serum DHEA, free testosterone, DHT and estradiol concentrations tended to be lower (P = 0.021) in athletes compared with non-athletes, but this was not statistically significant. In non-athletes, serum DHEA concentrations were significantly increased at all exercise intensities compared with baseline, whereas only maximal exercise induced a significant increase in serum DHEA concentrations for athletes (P < 0.01, F = , Fig. 1). Likewise, the serum free testosterone concentration was significantly increased at low-, moderate- and high-intensity exercise levels in non-athletes. However, in athletes, the serum free testosterone concentration was significantly increased only during high-intensity exercise (P < 0.01, F = ; Fig. 2). Additionally, in both the non-athlete and athlete groups, the serum DHT concentration increased significantly only with maximal

4 Exp Physiol (2016) pp Sex steroid hormone responses in athletes 171 Table 1. Subject characteristics Characteristic s (n = 8) s (n = 11) Age (years) 20.1 ± ± 0.9 Weight (kg) 61.3 ± ± 2.4 Height (cm) ± ± 2.4 Peak oxygen uptake 56.3 ± ± 2.1 (ml kg 1 min 1 ) Maximal heart rate (beats min 1 ) ± ± 2.4 P < 0.05 versus non-athletes. significant increase in the serum estradiol concentrations in both the non-athlete and athlete groups (Fig. 4). Cortisol concentration In non-athletes, the serum cortisol concentration was significantly increased at all exercise intensities compared with baseline, whereas only maximal exercise induced a significant increase in the serum cortisol concentration for athletes (P < 0.01, F = ; Fig. 5). exercise (P < 0.01, F = ; Fig. 3), and the serum DHEA and DHT concentrations in maximal exercise were higher in the athletes compared with the non-athletes. Moreover, moderate and maximal exercise induced a Discussion The present study is the first to demonstrate that systemic secretion of sex steroid hormones increases gradually in endurance athletes and that different Serum DHEA concentration (pg/ml) Figure 1. Dehydroepiandrosterone (DHEA) concentrations at each exercise intensity in athletes and non-athletes Abbreviations in this and subsequent figures: Pre, pre-exercise; 40%EX, exercise at 40% V O2 peak; 70%EX, exercise at 70% V O2 peak; and Max EX, exercise at 90% V O2 peak until exhaustion. Data are presented as means + SEM. P < 0.01 versus Pre; and P < 0.01 versus non-athletes. Serum free-testosterone concentration (pg/ml) Figure 2. Free testosterone concentrations at each exercise intensity in athletes and non-athletes Data are presented as means + SEM. P < 0.01 versus Pre.

5 172 K. Sato and others Exp Physiol (2016) pp Serum DHT concentration (pg/ml) Figure 3. 5α-Dihydrotestosterone (DHT) concentrations at each exercise intensity in athletes and non-athletes Data are presented as means + SEM. P < 0.01 versus Pre; and P < 0.01 versus non-athletes. Serum estradiol concentration (ng/ml) Figure 4. Estradiol concentrations at each exercise intensity in athletes and non-athletes Data are presented as means + SEM. P < 0.01 versus Pre. Serum cortisol concentration (μg/ml) Figure 5. Cortisol concentrations at each exercise intensity in athletes and non-athletes Data are presented as means + SEM. P < 0.01 versus Pre.

6 Exp Physiol (2016) pp Sex steroid hormone responses in athletes 173 exercise intensities cause different responses of sex steroid hormone concentrations in people with different levels of physical fitness. In non-athletes, all exercise intensity levels increased serum DHEA and free testosterone concentrations. On the contrary, in athletes, only high-intensity exercise caused a significant increase in serum DHEA and free testosterone concentrations. In both athletes and non-athletes, only high-intensity exercise caused a significant increase in serum DHT concentrations. Thus, the systemic secretion of sex steroid hormones in response to acute exercise may be elevated even at lower exercise intensity levels in non-athlete healthy young adults compared with athletes, whereas the secretion of sex steroid hormones, especially DHEA and DHT, during high-intensity exercise may be elevated in athletes immediately after exercise. In the present study, we demonstrated that only maximal-intensity exercise increased sex steroid hormone concentrations, especially DHEA and DHT concentrations, in adults with higher physical fitness levels immediately after each exercise session. On the contrary, Niemann et al. (2013) reported that saliva testosterone concentrations decreased in children (aged 9 10 years) with a low level of physical fitness in response to a single bout of exercise. Moreover, enzyme expressions related to circulating sex steroid hormone concentrations and muscle steroidogenesis tended to decrease at 3 h after moderate-intensity resistance exercise in our preliminary experiments on healthy young men. However, another study has demonstrated an increase in serum free testosterone and DHT concentrations immediately after repeated high-intensity aerobic exercise (consisting of 10 repetitions of 30 s, sprinting at a target load of 150% of the work capacity) in healthy active young men (Smith et al. 2013). In addition, high-intensity resistance exercise also increased testosterone concentrations during exercise compared with moderate-intensity exercise. Both aerobic and resistance exercise increased sex steroid hormone concentrations only during and immediately after exercise. The increase in the serum testosterone and DHT concentrations was short lived in younger individuals, returning to basal concentrations within 30 min to 1 h after exercise (Elias et al. 1993). In tissues such as muscle, brain and liver, testosterone, DHT and estradiol are synthesized by steroidogenesis-related enzymes, such as 17β-HSD, 3β-HSD, 5α-reductase and P450 aromatase from DHEA; therefore, the timing of sampling is important, because testosterone and DHT concentrations tend to decrease more than 1 h after exercise. Thus, a single bout of exercise may transiently elevate the secretion of sex steroid hormones, especially androgenic hormones, immediately postexercise. 5α-Dihydrotestosterone is generally synthesized from testosteronethrough 5α-reductase, which is an irreversible step in androgen metabolism, and a biophysiological action occurs when DHT binds to androgen receptors (Penning et al. 2000). In a previous study, we demonstrated that a single bout of moderate-intensity aerobic exercise increased muscle free testosterone and DHT concentrations and 5α-reductase enzyme expressions in normal healthy rats; also, acute aerobic exercise induced an increase in the free testosterone concentration (Aizawa et al. 2010). It might be suggested that an exercise-induced increase in circulating testosterone promotes DHT synthesis from testosterone in skeletal muscles. Additionally, the repeated elevation of DHT in response to the chronic day-to-day demands of an intense exercise programme may contribute to the adaptive responses of the future training load as well as muscle adaptation for athletes. Accordingly, to increase sex steroid hormones, high-intensity exercise would be needed for muscle adaption in athletes. However, muscle biopsies were not obtained in the present study; therefore, future studies should focus on the effect of acute exercise of different intensities on muscle steroidogenesis in people with different fitness levels. Systemic secretion of sex steroid hormones increased gradually with an increase in exercise intensity in the present study. In one of our previous studies, exercise-induced enhancement of Akt phosphorylation was suppressed by administration of an inhibitor that blocked the synthesis of DHT from testosterone (Sato et al. 2008, 2009). It was also reported that administration of testosterone increased satellite cell replication, inhibited satellite cell apoptosis and might cause muscle hypertrophy (Ferrando et al. 2002; Sattler et al. 2009). Additionally, testosterone deficiency induced impairment of mammalian target of rapamycin (mtor) and Akt phosphorylation, which might contribute to reduction of the skeletal muscle mass through downregulation of protein synthesis (Sinha-Hikim et al. 2006). However, the mechanisms underlying the acute exercise-induced elevation of sex steroid hormone secretion remain unclear. The test order in this experiment was not randomized in order to avoid acclimation to exercise, especially for non-athletes. Therefore, further studies should focus on the response of sex steroid hormone concentrations to exercise using a randomized test order as well as the timing of blood sampling to determine whether this has a different effect on sex steroid hormone concentrations. Further study is also needed to investigate the effect of acute exercise on the enzymes related to muscle sex steroid hormones and steroidogenesis to explain the exercise-induced changes in sex hormone steroidogenesis. Our previous studies demonstrated the gene and protein expressions of steroidogenic enzymes, such as 3β-HSD, 17β-HSD and 5α-reductase, in cultured muscle cells (Aizawa et al. 2007). We showed that muscle

7 174 K. Sato and others Exp Physiol (2016) pp steroidogenesis was enhanced by a single session of exercise (Aizawa et al. 2010) and that the exercise-induced change in sex steroid hormone concentrations has a sex difference in response to a single bout of exercise. In male rats, serum estradiol concentrations were increased only by acute aerobic exercise (Aizawa et al. 2008). In the present study, serum estradiol concentrations increased at moderate and high exercise intensities in both groups. Further study is needed on the effect of different exercise intensities on sex steroid hormone concentrations in women. Generally, the hypothalamic pituitary adrenal axis controls adrenal androgens; therefore, exercise could be more stressful for non-athletes. In fact, cortisol concentrations were significantly increased at low exercise intensities in non-athletes in the present study. Consequently, these results may indicate greater adrenal activation in non-athletes. Additionally, previous studies reported that serum testosterone concentrations were increased by progressive maximal-intensity exercise on a cycle ergometer, and the increases in luteinizing hormone (Brazeau et al. 2009) and follicle-stimulating hormone were synchronous with that of testosterone (Cumming et al. 1986). Luteinizing hormone and follicle-stimulating hormone are well known to stimulate testosterone synthesis and secretion by the gonads (Marshall et al. 1973). Moreover, acute aerobic exercise also increased serum corticotrophin-releasing hormone concentrations (Inder et al. 1998), and it is considered that exercise-induced increases in serum corticotrophin-releasing hormone concentrations may stimulate the adrenal cortex and induce sex steroid hormone secretion. However, the origin of the increased circulating sex steroid hormones caused by exercise remains unclear. Further study should focus on the hormones that directly stimulate sex steroid hormone secretion in the tissue. In summary, circulating sex steroid hormone responses differed between endurance athletes and non-athletes for different intensities of acute aerobic exercise, and the capacity for secretion of sex steroid hormones, especially DHEA and DHT, during high-intensity exercise may be enhanced in athletes. Therefore, only high-intensity exercise increased circulating DHEA and DHT concentrations in athletes, and these findings would contribute to exercise-induced physiological adaptation and to the maintenance a high performance level and athletic condition in athletes. References Aizawa K, Iemitsu M, Maeda S, Jesmin S, Otsuki T, Mowa CN, Miyauchi T & Mesaki N (2007). Expression of steroidogenic enzymes and synthesis of sex steroid hormones from DHEA in skeletal muscle of rats. Am J Physiol Endocrinol Metab 292, E577 E584. 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