Adipose tissue is not only a fat depot but is also recognized as an endocrine organ. It is capable of producing biologically active proteins called “adipokines” (11). These adipokines include adiponectin, leptin, tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), adipsin, and resistin (4). Adiponectin is a hormone secreted by adipocytes. It exhibits antiatherogenic and insulin-sensitizing properties (10). Adiponectin stimulates glucose use and fat oxidation in skeletal muscle (23) and has been implicated in the regulation of energy homeostasis (29). Adiponectin secretion is affected by many hormones such as insulin, catecholamines, and cortisol. They suppress adiponectin gene expression, and it has been demonstrated that exercise alters the concentration of these hormones (12). Both adiponectin and exercise increase substrate use and enhance insulin sensitivity (13). In addition to these effects of adiponectin, recent reviews and studies have focused on the antiinflammatory role of adiponectin, and in these studies (17,18), the anti-inflammatory potential of adiponectin is demonstrated.
IL-6 and TNF-α are important proinflammatory adipokines that are primarily secreted by the immune cells (2) and macrophages infiltrating adipose tissue (2,28), respectively. It has been demonstrated that muscle contractions induce a marked elevation in plasma IL-6 levels without any muscle damage (3,21). It has also been demonstrated that the release of IL-6 from exercising skeletal muscle is positively correlated to work intensity, glucose uptake, and plasma adrenaline concentration (7). In addition to the effects of exercise intensity, duration, and mode, it has been suggested that the exercise-induced increase in plasma IL-6 is related to the sympathoadrenal response to exercise (21).
Experimental studies have demonstrated that adiponectin expression is negatively regulated by proinflammatory cytokines including IL-6 and TNF-α, whereas adiponectin modulates the action and production of TNF-α in various tissues (18). However, no evidence suggests an association between plasma adiponectin and TNF-α levels in humans. Exercise elevates catabolic proinflammatory cytokines such as IL-6, IL-1, and TNF-α (5,16). Therefore, it was suggested that the assessment of the changes in these circulating mediators after different types of exercise training may assist in quantifying training loads (14).
Although the effects of anaerobic exercises on cytokine levels have been demonstrated in several studies (14,15), we could not find any study in the literature evaluating the effects of repeated Wingate tests (WTs) on plasma adiponectin, IL-6, and TNF-α levels. Furthermore, no study to date has investigated the relationship between the adiponectin and cytokine levels during the standardized model of anaerobic exercise (WT). Wingate test as a supramaximal exercise test involves the pedaling of a cycle ergometer for 30 seconds at a maximal speed against a resistance that is predetermined according to the subject's body weight (1). The WT strongly stimulates both the adenosine triphosphate-phosphocreatine and glycolytic systems (26) and thus activates purine catabolism and lactic acid production (6). In addition, supramaximal anaerobic exercise has been associated with a large increase in plasma catecholamine levels (30), which strongly affects adiponectin and cytokine levels. The Wingate test also represents a well-designed model for a single high-intense exercise bout (14).
We hypothesized that repeated bouts of supramaximal exercise may affect adiponectin and cytokine concentrations. Although exercise-induced changes in adiponectin level have been investigated in many studies (11,12), with different exercise protocols, no study to date has investigated the changes of adiponectin level in the postexercise period of the repeated bouts of the supramaximal exercise. Because anaerobic exercises affect several hormone levels such as those of insulin and cortisol (12), they may affect adiponectin levels as well. Furthermore, plasma cytokine levels may increase as a result of the repeated bouts of supramaximal exercise–induced catabolic environment in working muscles. Changes in plasma concentrations of various cytokines have been investigated during the postexercise period in many studies (14,15), but no information is available regarding the time course of changes in plasma levels of cytokines after repeated bouts of supramaximal exercise. Additionally, although an anti-inflammatory effect of adiponectin has been demonstrated in multiple studies (18), no study to date has investigated the changes of the postexercise adiponectin and cytokine levels and their relation with each other during the repeated bouts of supramaximal exercise. Therefore, this study was aimed to evaluate the effects of repeated bouts of supramaximal exercise on plasma adiponectin, IL-6, and TNF-α levels in sedentary men.
Experimental Approach to the Problem
The effects of repeated bouts of supramaximal exercise on adiponectin and inflammatory cytokines were studied in sedentary men. However, no study to date has investigated the effects of this type of anaerobic exercise on adiponectin and inflammatory cytokine levels. Therefore, 14 healthy and sedentary men performed 5 WTs with 2-minute intervals between tests. Blood samples were collected at preexercise, immediately after (within 1 minute), 15 minutes after, and 60 minutes after the fifth WT, and adiponectin, IL-6, and TNF-α levels were measured in these samples. In addition, we measured serum myoglobin levels, a commonly used marker for the assessment of muscle damage.
Fourteen healthy and sedentary men participated in the study. The physical characteristics of the participants are summarized in Table 1. All the participants were nonsmokers, and for at least 3 months before the study, they did not take any vitamins, minerals, or medications that would affect adiponectin and cytokine levels. The subjects were instructed not to change their physical activity, dietary habits, or any other aspects of their lifestyle during the study. Clinical examinations of the subjects were carried out during the study, and all the subjects were found to be healthy in this period.
The study protocol was approved by the Ethical Committee of Meram Faculty of Medicine, Selcuk University, Konya/Turkey. All the participants were informed about the purpose, procedures, and possible risks of the study before they signed a written informed consent.
Exercise tests started between 9 and 10 AM, 2 hours after a carbohydrate-rich, light breakfast, and consisted of five 30-second WT with a 2-minute rest between consecutive exercise bouts. Before the tests, each participant's body weight, height, and body fat percent were measured. The WT was performed on an Ergometric 894Ea Peak Bike (Monark Exercise AB, Varberg, Sweden) against a resistance of 75 g per kilogram of body weight (4.41 J per pedal revolution per kilogram of body weight). The optimal seat height was adjusted for each subject and was used for all WTs. The subjects' feet were firmly supported by toe clips and straps. The participants were allowed to pedal unloaded and instructed to reach a pedaling rate of 100 rpm. The predetermined load was applied to the flywheel automatically by the Monark Anaerobic Test Software, and the participants were verbally encouraged to maintain as high a pedaling rate as possible throughout the 30-second test duration. The subjects rested on the bicycle ergometer for an interval of 2 minutes between subsequent tests.
Each participant was asked to lie down, and a catheter was inserted into an antecubital vein. Four blood samples of 10 ml were collected (a) at preexercise, (b) immediately after (within 1 minute), (c) 15 minutes after, and (4) 60 minutes after the fifth WT. Ethylenediaminetetraacetic acid coated tubes were used for the analysis of plasma adiponectin, IL-6, and TNF-α. Plasma was obtained by the centrifugation of whole blood at 3,000 rpm for 10 minutes. Blood used for analysis of myoglobin was collected into tubes containing no additive, allowed to clot at room temperature for 30 minutes, and then the serum was separated by centrifugation at 3,000 rpm for 10 minutes. The samples were stored at −80°C until the time of the analysis.
The quantitative measurements of human plasma adiponectin, IL-6, and TNF-α levels were determined using an enzyme-linked immunosorbent assay (Bio-Tek ELX800 Bio-Tek Instruments Inc., Winooski, VT, USA) using commercially available kits (Biovendor Laboratory Medicine Inc., Brno, Czech Republic, and IBL Immuno Biological Laboratories, Hamburg, Germany, respectively) according to the manufacturer's instructions. Adiponectin, IL-6, and TNF-α levels were expressed in micrograms per milliliter, picograms per milliliter, and picograms per milliliter, respectively.
Serum myoglobin concentration was measured using automated chemiluminescence methods by using commercially available myoglobin kits (Unicel DXI 800 instrumentation; Beckman Coulter, Fullerton, CA, USA) and levels were expressed in nanograms per milliliter. Plasma volume shifts were not corrected because similar studies (14) did not apply to this method.
Statistical analysis was performed with SPSS 15.0 for Windows. All data are presented as mean ± SD. The distribution of variables was evaluated using the Shapiro-Wilk test. Repeated measures analysis of variance was used to assess the effects of exercise on adiponectin levels in plasma with the time serving as the within-group factor followed by t-tests, with the Bonferroni correction for multiple comparisons because adiponectin levels showed normal distribution. The IL-6, TNF-α and myoglobin levels were analyzed with the Friedman test and Wilcoxon signed-rank test with Bonferroni correction, as they did not show normal distribution. A p value ≤ 0.05 was considered to be statistically significant.
The effects of the repeated bouts of supramaximal exercise on adiponectin, IL-6, TNF-α, and myoglobin levels are given in Table 2. Plasma adiponectin levels decreased in serum samples taken 60 minutes postexercise (36.7 ± 2.2 μg·mL−1) compared with preexercise (37.9 ± 2.1 μg·mL−1) and immediately postexercise (38.1 ± 3.1 μg·mL−1) (p < 0.05). Plasma IL-6 levels increased immediately (118.9 ± 41.8 pg·mL−1), 15 minutes (117.5 ± 36.1 pg·mL−1), and 60 minutes (133.8 ± 34.1 pg·mL−1) postexercise compared with preexercise (100.9 ± 13.0 pg·mL−1) (p < 0.05). Additionally, plasma IL-6 levels increased 60 minutes postexercise compared with the 15 minutes postexercise (p < 0.05). Plasma TNF-αlevels did not change with the supramaximal exercises compared with preexercise (p > 0.05). Serum myoglobin levels did not change with the supramaximal exercise (p > 0.05).
The main finding of this study was a marked decrease in adiponectin levels and a marked increase in IL-6 levels in the postexercise period of the repeated bouts of the supramaximal exercise. Exercise-induced changes in adiponectin and inflammatory cytokine levels have been investigated in many studies (11,12) with different exercise protocols. However, no study to date has investigated the effects of repeated bouts of supramaximal exercise—one of the most commonly used exercise test to assess anaerobic power—on adiponectin and inflammatory cytokine levels.
In this study, adiponectin levels decreased, whereas IL-6 levels increased in the postexercise period compared with that in the preexercise period. However, it is observed that TNF-α and myoglobin levels did not change. There are contradictory results in the literature about the effect of exercise on adiponectin levels. It has been claimed that acute exercises would not affect (4,12) or cause an increase (9) in plasma adiponectin levels compared with that preexercise. Kraemer et al. (12) showed that acute exercise did not affect circulating adiponectin levels. Ferguson et al. (4) found that adiponectin did not change with cycling exercise in healthy men and women. In contrast to these results, Jürimäe et al. (9) showed a 20% increase in adiponectin levels 30 minutes after acute intense exercise. It has been suggested that high circulating adiponectin would be beneficial during exercise, because adiponectin favors fat oxidation and uptake of glucose into skeletal muscle facilitating the production of energy by aerobic metabolism (25). All the investigations mentioned above evaluated the changes in adiponectin concentration in response to aerobic exercise. We could not find any study investigating the effects of anaerobic exercises on adiponectin concentrations. In our study, adiponectin levels decreased 60 minutes postexercise compared with that preexercise and immediately postexercise. These findings suggest that decreased adiponectin levels may be because of the increase of the IL-6 levels. Because it has been reported that proinflammatory cytokines suppress adiponectin expression in adipocytes (18) and exercise caused to increase in IL-6 levels. Consistent with the findings of previous reports, in this study, IL-6 levels significantly increased in the postexercise period. Recent studies clearly demonstrate that muscle contractions without any muscle damage induce a marked elevation in plasma IL-6 levels (3,14,20,22). The major source for the exercise-related IL-6 increase is the skeletal muscle (5). In addition to the effects of exercise intensity, duration, and mode, it has also been suggested that the exercise-induced increase in plasma IL-6 is related to the sympathoadrenal response to exercise (22). However, IL-6 is believed to play an important mediatory role in the inflammatory response needed for the exercise-associated muscle damage repair (21,27). This may explain why, in this study, the levels of IL-6 remained elevated during the recovery period from the anaerobic-type exercise as well. Our finding of an increase only in IL-6 levels suggests that IL-6 is probably the most sensitive inflammatory cytokine to exercise or that anaerobic exercise may lead to a different hormonal catabolic environment. It has been reported that both the eccentric and concentric contractions resulted in elevated levels of IL-6 mRNA locally in the exercised muscle, whereas the level in resting muscle was not elevated (8). It appears, therefore, that IL-6 production is associated with contracting muscle and is not a systemic effect. It has been demonstrated that the release of IL-6 from working skeletal muscle is positively related to work intensity, glucose uptake, and plasma adrenaline concentration (7).
In this study, the TNF-α level did not change with repeated bouts of supramaximal exercise. In several studies (3,22), it has been reported that exercise did not induce an increase in plasma levels of TNF-α. However, Rhind et al. (24) showed that a single bout of anaerobic exercise increased circulating TNF-α levels by 83%. The difference in the results may depend on the exercise intensity, duration, and mode. In our study, during the postexercise period, unchanged TNF-α levels may depend on the inhibitory role of IL-6, because, it has been demonstrated that IL-6 inhibits TNF-α production and TNF α–induced insulin resistance (21).
Myoglobin is a commonly used marker of skeletal muscle damage. It presents a proxy marker of damage to the muscle cell membrane (19). In this study, serum myoglobin concentration did not change with the repeated bouts of supramaximal exercise. These results demonstrated that this type of exercise did not cause muscle damage. The observed changes in IL-6 levels cannot be attributed to muscle damage.
In conclusion, repeated bouts of supramaximal exercise caused a decrease in adiponectin levels and caused an increase in IL-6 levels. Decreased adiponectin levels with the repeated bouts of supramaximal exercise may depend on its antiinflammatory role. Repeated bouts of supramaximal exercise do not induce muscle damage, but they cause an inflammatory response in exercised muscle and increase plasma IL-6 levels. More detailed investigations are needed to explain these changes.
This study has examined the hormonal and inflammatory responses to repeated bouts of supramaximal exercises. This study suggests that repeated bouts of supramaximal exercises cause an increase in inflammatory cytokine levels and a decrease in adiponectin levels. According to our results, repeated supramaximal exercises induce an inflammatory response, and this inflammatory response inhibits adiponectin secretion. This type of exercises may decrease substrate use and attenuate insulin sensitivity. However, the response of these cytokines and hormone to different types and durations of exercises can be studied by athletes and coaches and may be used as an objective tool to determine the load and duration of the exercise and to plan better exercise cycles during the training sessions. Also, further research is needed to determine the mechanism of action in this type of exercises in athletes.
The authors thank Professor Said Bodur for statistical assistance and Çiğdem Dölek, Safiye Sayar, and Emrah Uğur for technical assistance. There is no grant support for this study.
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