It is well known that resistance exercise training elicits morphological and functional adaptations in the skeletal muscle, namely skeletal muscle hypertrophy and increased muscle strength (14,23,27). Degrees of the muscular hypertrophy and strength gains after resistance training are thought to be dependent on the intensity of exercise, in such a way that an intensity of more than 65% of the maximum strength [one-repetition maximum (1 RM)] is required to achieve a substantial effect (22).
It is suggested that combinations of multiple factors, including mechanical, metabolic, endocrine, and neural factors, are involved in muscular hypertrophy after resistance training. Among these factors, some anabolic hormones such as growth hormone (GH) and testosterone are secreted in response to resistance exercise and play important roles in promoting muscular hypertrophy (8). McCall et al. (21) have demonstrated that resistance exercise-induced GH response was highly correlated with the magnitude of muscle hypertrophy. A previous study has shown that moderate-intensity resistance exercise using muscular hypertrophy protocol (10 RM with 1-min rest intervals) considerably elevated GH and testosterone but not those using higher intensity (5 RM) and a longer rest period (3 min) (19).
It has been shown that anabolic hormone secretions from the hypothalamus-pituitary may be stimulated by the local accumulation of metabolic subproducts such as lactate and hydrogen ions (11,12,14,29,34). Gordon et al. (11) have demonstrated that alkalosis treatment by sodium bicarbonate (NaHCO3) ingestion resulted in greater pH and attenuated GH response to exercise. In addition, Goto et al. (12) have indicated that resistance exercise-induced metabolic stress was associated with acute GH response but not testosterone. Although the mechanism by which anabolic hormone secretions, especially GH, are stimulated by acid-base changes is yet to be fully understood, it is hypothesized that exercise with greater metabolic stress may cause greater GH response to resistance exercise.
Several recent studies have shown that low-intensity resistance exercise combined with moderate vascular occlusion effectively causes increases in muscular size and strength (20,30-32). It has also been reported that low-intensity exercise combined with occlusion markedly enhanced metabolite accumulation within the muscles and concomitant GH secretion (29). Thus, researchers have speculated that local hypoxia by the vascular occlusion may contribute to these adaptations, but the underlining mechanism is still not clarified. Moreover, to date, there are no data that describe the impact of acute systemic hypoxia on metabolic and anabolic hormone responses to resistance exercise.
Here, we examined the effects of resistance exercise on metabolic and hormonal responses under acute systemic hypoxia. We hypothesized that the resistance exercise in the hypoxic condition would cause greater accumulation of metabolic subproducts and greater responses of anabolic hormones.
Twelve healthy, nonsmoking, male subjects (age = 29.9 ± 1.2 yr, height = 172.4 ± 1.2 cm, body mass = 68.9 ± 2.0, bench press 1 RM = 80.5 ± 5.0 kg, bilateral leg press 1 RM = 177.8 ± 7.4 kg) participated in this study. All subjects were physically active and had experience with recreational exercise training. However, none of the subjects was involved in any regular training program at the beginning of the study. Before or during participation in this study, subjects reported no exposure to an altitude of >3000 m within 1 month before each trial, no history of severe acute mountain sickness, and no medications (e.g., anabolic steroids, creatine, sympathoadrenal drugs) taken during the experimental trials. They were informed about the experimental procedure as well as the purpose of the present study, and their written informed consent was obtained in advance. The study was approved by Japan Institute of Sports Sciences Ethics Committee.
The subjects visited the laboratory before the experimental trials. The 1 RM for bench press and bilateral leg press exercises were measured using weight-stack machines under room air normoxic condition. Before 1 RM was assessed, subjects were invited to stretch for several minutes; they then performed two warm-up sets of 10 repetitions. The load was increased until the subjects were unable to perform a lift. At least 3 min of rest was set between each trial to avoid fatigue. Three to five trials were conducted to determine 1 RM.
The study was conducted using a single-blinded crossover design. All subjects participated in two experimental trials separated by more than 4 wk in random order: 1) resistance exercise while breathing room air [normoxic resistance exercise (NR)] and 2) resistance exercise while breathing 13% oxygen [hypoxic resistance exercise (HR)] using a hypoxic generator (HYP-100; Hypoxico, Inc., New York, NY). The resistance exercise consisted of two consecutive exercises (bench press and bilateral leg press), each with 10 repetitions for five sets at 70% of 1 RM. The subjects were allowed to rest for 1 min between all the sets and exercises. Subjects were instructed to lift and lower the load at a constant velocity, taking approximately 2 s for each repetition. If the load became too heavy, the subject was assisted. The range of motion in each set of exercises was from 90° to 0° (0° at full extension). The trials were performed between 08:00 and 11:30 a.m. to avoid diurnal variations in metabolism and hormonal responses.
Measurement of muscle oxygenation during exercises.
Local muscle oxygenation profiles of the left vastus lateralis muscle during exercises (bilateral leg press) were made with a near-infrared spectroscopy (Hamamatsu NIRO 300; Hamamatsu Photonics KK, Hamamatsu, Japan). The wavelengths of emission light were 775, 810, 850, and 910 nm, and the relative concentrations of oxygenated hemoglobin (Oxy-Hb) in tissues were quantified using the modified Beer-Lambert law (39). The near-infrared spectroscopy signals registered during exercise do not reflect the absolute levels of oxygenation. Therefore, the changes of oxygenation in muscles are expressed as relative values to the overall changes in the signal monitored according to the arterial occlusion method (4). The resting level (normoxic condition) of Oxy-Hb was regarded as 100% (baseline), and the minimum plateau level of Oxy-Hb (by arterial occlusion) was regarded as 0%. A pressure cuff was placed around the proximal area of the left thigh and inflated to more than 300 mm Hg until the minimum plateau level of Oxy-Hb appeared. The distance between the incident point and the detector was 30 mm.
Blood sampling and analyses.
After an overnight fast, the subjects came to the laboratory and rested for 30 min before the first blood collection. Venous blood samples were obtained from each subject's forearm before normoxia and hypoxia exposures (pre 1), 15 min after normoxia and hypoxia exposures (pre 2), and at 0 (immediately after the exercise), 15, 30, and 60 min after exercise. The exposures were continued until experimental trials ended (60 min after exercises). Serum and plasma samples were separated from blood cells by centrifugation (3000 rpm for 15 min) and stored at −80°C until analysis. Serum GH concentration was measured by immunoradiometric assay using a commercial kit (TFB, Inc., Tokyo, Japan). Concentrations of plasma epinephrine (E) and norepinephrine (NE) were measured with high-performance liquid chromatography using a commercial kit (Tosoh Co., Tokyo, Japan). Serum insulin-like growth factor 1 (IGF-1) was determined by immunoradiometric assay using a commercial kit (Mitsubishi Chemical Medience Co., Tokyo, Japan). Serum testosterone concentration was measured by radioimmunoassay using a commercial kit (DPC Co., Tokyo, Japan). Serum cortisol concentration was measured by radioimmunoassay using a commercial kit (TFB, Inc.). Blood samples were also obtained from the fingertip to measure lactate concentration using automatic lactate analyzer (Lactate Pro; ARKRAY, Kyoto, Japan). Arterial oxygen saturation (SpO2) was measured by pulse oximetry from the second finger (PULSOX-Me300; Tenjin Ltd., Osaka, Japan).
All data except for muscle oxygenation during exercises were analyzed by a two-way ANOVA with repeated measures using Stat View 5.0 (Hulinks, Tokyo, Japan). If significant differences existed, a post hoc analysis test (Bonferroni/Dunn) was performed. The mean values of metabolite, hormone, and catecholamine concentrations after exercises between the trials were compared using paired t-tests. Paired t-test was used to compare the mean values of minimum muscle oxygenation level during each set of exercises between trials. The level statistical significance was set at P < 0.05. Data are expressed as mean ± SE.
Arterial oxygen saturation.
Figure 1 shows SpO2 measured before and after the exercises. SpO2 in the HR trial was lower than that in the NR trial (ANOVA, P < 0.01). In the NR trial, SpO2 significantly decreased at 0 min after the exercise compared with the pre 1 value (P < 0.01). In contrast, SpO2 in the HR trial was significantly lower at all time points after hypoxia exposure (from pre 2 to 60 min after exercise) than the pre 1 value (P < 0.01). In addition, SpO2 in the HR trial was lower than that in the NR trial at all time points after hypoxia exposure (P < 0.01).
Figure 2 shows the mean values of minimum oxygenation level in the left vastus lateralis muscle during each set of exercises. The mean value of minimum oxygenation level in the HR trial (17.1% ± 2.9% baseline) was significantly lower than that in the NR trial (27.2% ± 2.1% baseline; P < 0.05).
Blood lactate and GH concentrations.
Figure 3 shows blood lactate and serum GH concentrations measured before and after the exercises. There were no significant differences in blood lactate and serum GH between before (pre 1) and after (pre 2) the exposures in either trial. Blood lactate concentration in the HR trial was higher than that in the NR trial (ANOVA, P < 0.05). In the NR trial, blood lactate concentration significantly increased at 0, 15, and 30 min after exercise compared with the pre 2 value (P < 0.01). Meanwhile, blood lactate concentration at all time points after the exercise was significantly higher than that at the pre 2 value in the HR trial (from 0 to 30 min after exercise, P < 0.01; 60 min after exercise, P < 0.05). In addition, the mean values of blood lactate concentration after exercises were significantly higher in the HR trial (5.7 ± 0.4 mmol·L−1) than in the NR trial (4.6 ± 0.4 mmol·L−1, P < 0.01). Serum GH concentration did not significantly change in the NR trial. On the other hand, serum GH concentration in the HR trial was significantly increased at 15 and 30 min after exercise compared with the pre 2 value (P < 0.05). In addition, the mean value of GH after the HR trial (12.9 ± 2.5 ng·mL−1) was significantly higher than that after the NR trial (7.7 ± 1.9 ng·mL−1, P < 0.01).
E and NE concentrations.
Figure 4 shows plasma E and NE concentrations measured before and after the exercises. There were no significant differences in plasma E and NE between before (pre 1) and after (pre 2) the exposures in both the trials. In both the NR and HR trials, plasma E and NE concentrations significantly increased at 0 min after the exercises compared with the pre 2 values (P < 0.01). In contrast, the mean value of E after the HR trial (0.06 ± 0.04 ng·mL−1) was significantly higher than that after the NR trial (0.04 ± 0.04 ng·mL−1, P < 0.01). The average values of NE after exercises were also significantly higher in the HR trial (0.72 ± 0.08 ng·mL−1) than those in the NR trial (0.61 ± 0.06 ng·mL−1, P < 0.05).
IGF-1, testosterone, and cortisol concentrations.
Table 1 shows serum IGF-1, testosterone, and cortisol concentrations measured before and after the exercises. There were no significant differences in serum IGF-1, testosterone, and cortisol between before (pre 1) and after (pre 2) the exposures in both the trials. In both the NR and HR trials, serum IGF-1 concentrations significantly increased at 0 min after the exercises compared with the pre 2 values (P < 0.01). Serum testosterone concentrations were also significantly increased at 0 min after exercises compared with the pre 2 values in both the NR (P < 0.05) and the HR (P < 0.01) trials. Serum cortisol concentration did not significantly change in the NR trial. In contrast, serum cortisol concentration in the HR trial was significantly increased at 15 and 30 min after exercise compared with the pre 2 value (P < 0.01).
In the present study, we demonstrated that resistance exercise in hypoxic condition (HR) caused larger elevations of blood lactate, GH, E, and NE concentrations than that in normoxic condition (NR). The SpO2 in the HR trial (mean value after exposure: 84.1% ± 0.6%) was significantly lower than that in the NR trial (mean value after exposure: 97.0% ± 0.2%). The mean value of minimum muscle oxygenation level during exercise in the HR trial was also significantly lower than that in the NR trial. These results suggest that resistance exercise in acute systemic hypoxia evokes greater accumulation of metabolic subproducts and hormonal responses.
The peak concentration of blood lactate after the HR trial was 1.2-fold higher than that of NR trial (Fig. 3). This greater lactate production in the HR trial was presumably caused by hypoxia. Because samples were taken from circulating blood, the local concentration of lactate within the exercising muscle is likely much higher. Previous studies have demonstrated that resistance exercise with greater metabolic stress caused greater GH and catecholamine responses (12) and ischemic exercise with increased accumulation of metabolite caused strong GH and catecholamine responses (29). In the present study, the mean values of GH (1.7-fold), E (1.5-fold), and NE (1.2-fold) secretions after the HR trial were significantly higher than those in the NR trial (Figs. 3 and 4). Therefore, the greater metabolic accumulation caused by resistance exercise in hypoxic condition might have induced the elevations of GH and catecholamine in the HR trial. In addition, it has been reported that catecholamine could directly stimulate GH secretion from pituitary tissue in vitro (10) and heighten E and NE outflow implicated as a possible moderator of exercise-induced GH release (38). The larger increases in E and NE concentrations might also contribute to the greater GH response in the HR trial.
Several studies have reported that low-intensity resistance training combined with vascular occlusion caused muscular hypertrophy in humans (20,30-32). However, the detailed mechanism of the muscle hypertrophy by low-intensity resistance exercise with occlusion has not been understood, although the greater GH secretion have been implicated as a cause of the low-intensity resistance training with occlusion-induced muscular hypertrophy (9,20,29,30,32). In the present study, we revealed that systemic hypoxia was actually associated with greater GH response to resistance exercise for the first time. The hypoxia may play a key role in the low-intensity resistance training with vascular occlusion-induced muscular hypertrophy.
IGF-1 and GH play various physiological roles such as cell growth and maintenance, in skeletal muscle. It is reported that moderate-resistance exercise (10 RM with 1-min rest intervals) increases serum concentration of IGF-1 as well as GH (17). In the present study, we also showed that serum IGF-1 concentrations increased immediately after both NR and HR trials (Table 1). The magnitude of changes in GH after the exercises was quite different between trials, and the GH was significantly increased at 15 and 30 min only after the HR trial (Fig. 3). However, it is reported that the synthesis and secretion of IGF-1 are stimulated by GH (6). Therefore, the increases in circulating IGF-1 immediately after both trials in this study might be GH-independent.
To date, many researchers have reported that resistance exercise induced acute increases in serum level of testosterone (1,7,14,19,26). The present study also showed that the serum testosterone levels increased immediately after the resistance exercises in both conditions (Table 1). The serum testosterone responses after the exercises were not different between the trials as opposed to lactate and GH responses in the present study. Kraemer et al. (19) demonstrated that serum testosterone increased after resistance exercise without accumulation of metabolic subproduct (blood lactate). Fujita et al. (9) reported that serum testosterone did not change after low-intensity ischemic resistance exercise, although metabolic stress markedly increased after the exercise. Moreover, previous studies showed that serum testosterone responses were not affected by the degree of metabolic stress after resistance exercise (7,12). These results suggest that resistance exercise-induced metabolic changes do not affect serum testosterone response, implying that other factors such as muscle mass (15,38) and intensity and volume (3,13,19,24) of resistance exercise might be associated with the testosterone response.
Cortisol is released from the adrenal cortex in response to the stresses during exercise. Previous studies have shown significant elevations in cortisol after resistance exercise (9,17,18). Several researchers have demonstrated that acute cortisol response to resistance exercise might depend on total exercise stress and metabolic requirements (12,26). The increased metabolic requirements in hypoxic condition might cause the increase in serum cortisol in the HR trial. This mechanism is speculative, and further studies are needed to clarify the basic mechanisms of cortisol release induced by resistance exercise in systemic hypoxia.
In the present study, GH in both trials, blood lactate, and two hormones (E and cortisol) in the HR trial did not return to baseline values within 60 min after exercise. It may be necessary to measure these parameters at later time points after resistance exercises to clarify the effects of acute hypoxic resistance exercise on metabolic and hormonal responses in the future.
We also showed that there were no differences in all measurement items except SpO2 between before and 15 min after the hypoxic exposure (before exercise). These results suggest that metabolite accumulations and hormonal responses are unaffected only by acute hypoxia. These results (blood lactate, GH, and cortisol) are consistent with the results of a previous study (28).
So far, some researchers have investigated muscular adaptations to aerobic exercise training in hypoxic condition (2,5,33,35,40). They have reported that hypoxic training resulted in greater increase in skeletal muscle capillarity (5), myoglobin (33), mitochondrial volume (35), and messenger RNA expressions involving muscular endurance (35,40), although there is controversy regarding the aerobic exercise training in hypoxic condition-induced mitochondrial enzyme activity in skeletal muscle (2,33). These results suggest that hypoxic exposure is one of the important factors in aerobic training-induced muscular adaptations. In contrast, the effect of resistance exercise training under systemic hypoxia on muscular adaptations is completely unknown. It has been reported that the venous occlusion for 2 wk increased expressions of genes involving muscular hypertrophy in rats (16), but a single bout of low-intensity resistance exercise combined with occlusion did not change in humans (6). Therefore, it is necessary to investigate whether hypoxic exposure plays an important role for the expressions of genes involving muscular hypertrophy in the future.
In conclusion, we demonstrated that resistance exercise in acute systemic hypoxia caused greater accumulation of lactate and greater response in GH, E, and NE to a bout of resistance exercise. Our data suggest that hypoxia is a potent factor for the enhancements of anabolic hormone (GH) response to resistance exercise.
The authors thank members of the hypoxic research project at the Japan Institute of Sports Sciences for their critical comments and excellent technical support. The authors also thank Miki Hirashima, Ayako Hasegawa, Ayano Naka, and Yoshie Tanaka for their help with the conduct of the clinical portion of this study. This work was supported by grants-in-aid for Scientific Research from the Japan Institute of Sports Sciences. The results of the present study do not constitute endorsement by the American College of Sports Medicine.
M.K., T.I., and T.A. were responsible for the study concept and design, acquisition of subjects and data, analysis and interpretation of data, and preparation of manuscript. T.H. and Y.S. were responsible for the acquisition of subjects and data and analysis and interpretation of data. T.K. was responsible for the acquisition of subjects and data, for obtaining funding, and for supervision. None of the authors had a conflict of interest.
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