Physical inactivity and a sedentary lifestyle are highly associated with the development of a low-grade chronic inflammation state that is linked to several medical conditions such as obesity, insulin resistance, type 2 diabetes mellitus, cardiovascular and chronic obstructive pulmonary diseases, and others (14). In contrast, regular physical exercise has anti-inflammatory effects and can act on the prevention and/or treatment of the mentioned conditions (14). It is suggested that “exercise is medicine” (22), but increased amount of exercise is not always the best option. For example, athletes normally used high-intensity training sessions to enhance and maintain performance. However, this strategy may lead to athletes’ temporary performance decrease and acute fatigue (16). Thus, an adequate recovery period is essential to athletes achieving supercompensation and improving their performance.
The imbalance between training and recovery may lead to overreaching (OR) and/or overtraining syndrome (29). About overtraining (OT) animal models, Pereira et al. (36) developed an 8-wk protocol based on eccentric exercise (EE) sessions. The authors verified that Swiss mice did not restore their performance even after 2 wk of total recovery (36) and concluded that the protocol led to nonfunctional overreaching, a performance decrement that can be reversed after weeks or months of recovery and may be associated with psychological and hormonal disturbances (29). The overtrained mice also presented low-grade chronic inflammation in muscle (i.e., high protein levels of interleukin 6 [IL-6] and tumor necrosis factor α [TNF-α]) and in serum (i.e., high levels of IL-6) as well as high protein levels of myostatin in muscle samples (37).
High levels of IL-6, TNF-α, and myostatin are related to the impairment of insulin signaling pathway and insulin resistance (15,19,44); however, the relationship between EE and these metabolic disorders is not clear (10,11,24,32,33). The muscle damage induced by EE may include inflammation leading to high serum levels of TNF-α, and not IL-6 or IL-1β (9), that increase insulin receptor substrate 1 (IRS-1) serine phosphorylation and decrease the association between IRS-1 and phosphoinositide 3-kinase (PI3-k) and protein kinase B (Akt) serine phosphorylation, impairing the insulin signaling pathway (25). Considering the studies of Kirwan and del Aguila (25) and Pereira et al. (37), the first aim of the present investigation was to verify the effects of the OT protocol (36) on the protein contents of IRS-1, phospho (p)-IRS-1 (Ser307), insulin receptor beta (IRbeta), pIRbeta (Tyr1146), and Akt and pAkt (Ser473) in skeletal muscles with the different fiber type compositions of Swiss mice. Our hypothesis is that chronic EE is linked to insulin signaling impairment.
In addition, other proteins such as the IκB kinase (IKK), an enzymatic complex linked to the cellular response to inflammation; the stress-activated protein kinases/Jun amino-terminal kinases (SAPK/JNK), a member of the mitogen-activate protein kinase family; and the suppressor of cytokine signaling 3 (SOCS3), a member of the SOCS family, are related to the impairment of the skeletal muscle insulin signaling (7,23,38,39,42). Thus, our second aim was to verify whether the OT protocol modulates the protein contents of IKKalpha/beta, pIKKalpha/beta (Ser176/180), SAPK/JNK, pSAPK/JNK (Thr183/Tyr185), and SOCS3 in skeletal muscles with the different fiber type compositions of Swiss mice.
Male Swiss mice from the Central Animal Facility of the Ribeirão Preto campus of the University of Sao Paulo were maintained in individual cages with controlled temperature (22°C ± 2°C) on a 12-h light–12-h dark inverted cycle (light: 6:00 p.m. to 6:00 a.m.; dark: 6:00 a.m. to 6:00 p.m.) with food (Purina chow) and water ad libitum. The experimental procedures were approved by the ethics committee of the University of Sao Paulo and adhere to the American College of Sports Medicine’s animal care standards. Eight-week-old Swiss mice were divided into three groups: control (C; sedentary mice; n = 12), trained (TR; performed the aerobic training protocol; n = 12), and overtrained (OTR; performed the OT protocol; n = 12). The C, TR, and OTR mice were manipulated and/or trained in a dark room between 6:00 and 8:00 a.m. (36).
Incremental load test
Mice were adapted to treadmill running (INSIGHT®; Ribeirão Preto, São Paulo, Brazil) for 5 d, for 10 min·d−1 at 3 m·min−1 (36,37). As previously described (13), rodents performed the incremental load test with an initial intensity of 6 m·min−1 at 0%, with increasing increments of 3 m·min−1 every 3 min until exhaustion, which was defined when mice touched the end of the treadmill for five times in 1 min. Mice were encouraged using physical prodding. The exhaustion velocity (m·min−1) of mice was used to prescribe the intensities of aerobic training and OT protocols.
Aerobic training protocol
As previously described (36,37), the 8-wk aerobic training protocol was based on the study of Ferreira et al. (13), and each experimental week consisted of 5 d of training followed by 2 d of recovery.
As previously described (37), the 8-wk OT protocol was based on the study of Pereira et al. (36), and each experimental week consisted of 5 d of training followed by 2 d of recovery. During the first 4 wk of OT protocol (i.e., first stage), the volume was gradually increased to reach 60 min·d−1 in the fourth week. In this first stage, the rodents ran at a grade of 0%. In the fifth week of OT protocol, the volume was maintained, but rodents ran at a grade of −14%. This running grade was maintained until the end of OT protocol. In the sixth and seventh weeks of OT protocol, the intensity and volume increased to 25% and 50% compared with the fifth week, respectively. In the eighth week of the OT protocol, the number of daily sessions was doubled compared with the seventh week. The rest interval between the daily sessions was 4 h.
The incremental load test (i.e., exhaustion velocity) and the exhaustive test (i.e., time to exhaustion) were used as performance evaluation parameters 24 and 48 h after the last training session (i.e., aerobic training or OT sessions), respectively. The incremental load test was performed on week 0 and at the end of week 8. Blood samples were taken from the tails of mice using 25-μL heparinized capillary tubes before, immediately after, and at the third and fifth minute after the incremental load test performed at the end of week 8. Blood lactate concentrations (mM) were assayed by a lactate analyzer (YSI 1500 Sport; Yellow Spring Instruments, OH). As previously described (36,37), because of the high intensity and treadmill inclination, the exhaustive test was performed at the end of weeks 4 and 8.
As previously described (36,37), 24 h after the incremental load test, the rodents ran at 36 m·min−1 with 8% treadmill grade until exhaustion, which was defined as when mice touched the end of treadmill five times in 1 min. Mice were encouraged using physical prodding. This value was recorded as the time to exhaustion (s).
Metabolic parameters and insulin tolerance test
The body weight and the food intake of the experimental groups were recorded weekly. Food intake was determined by subtracting by the final food weight (i.e., weight of food put in each individual cage after 1 wk) from the initial food weight (i.e., weight of food put in each individual cage on Monday morning), as previously described (36,37). At the end of week 7, 48 h after the last aerobic training or OT sessions, fed mice were injected intraperitoneally with human recombinant insulin (1.5 U·kg−1, Eli Lilly, Indianapolis, IN). Blood samples from tails were collected at 0, 5, 10, 15, 20, 25, and 30 min to measure blood glucose concentrations using a glucometer (Accu-chek; Roche Diagnostic Corp., Indianapolis, IN). The area under the insulin curves were calculated by the trapezoidal principle (28).
Muscle and total blood collections
Mice were anesthetized 24 h after the exhaustive test (i.e., at the end of week 8). After an overnight fast (∼12 h), rodents were anesthetized with an intraperitoneal (i.p.) injection of 2,2,2-tribromoethanol 2.5% (10–20 μL·g−1). As soon as anesthesia was ensured by the loss of pedal and corneal reflexes, mice were injected intraperitoneally with saline or saline with human recombinant insulin (10 U·kg−1; Eli Lilly, Indianapolis, IN). After 10 min, the extensor digitorum longus (EDL) and soleus muscles of both hindlimbs were removed and stored at −80°C for subsequent protein analysis by immunoblotting.
Protein analysis by immunoblotting
The EDL and the soleus muscle samples were ablated, pooled (i.e., muscles sample from a single animal were used to prepare a single lysate), minced coarsely, and homogenized in extraction buffer (1% Triton X-100, 100 mM Tris, pH 7.4, containing 100 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM EDTA, 10 mM sodium vanadate, 2 mM PMSF, and 0.1 mg·mL−1 aprotinin) at 4°C with a Polytron PTA 20S generator (Brinkmann Instruments model PT 10/35), operated at maximum speed for 30 s. The extracts were centrifuged (9900g) for 40 min at 4°C to remove insoluble material, and the supernatants of these homogenates were used for protein quantification using the Bradford (4) method.
Proteins were denatured by boiling in Laemmli sample buffer containing 100 mM DTT, run on SDS–PAGE gel, and transferred to nitrocellulose membranes (GE Healthcare, Hybond ECL, RPN303D). The transfer efficiency to nitrocellulose membranes was verified by brief staining of the blots with Ponceau red stain. These membranes were then blocked with Tris-buffered saline containing 5% BSA and 0.1% Tween-20 for 1 h at 4°C. The antibodies used for immunoblotting overnight at 4°C were insulin receptor β (4B8) Rabbit mAb (no. 3025), phospho-IGF-I receptor β (Tyr1131)/insulin receptor β (Tyr1146) (no. 3021), IRS-1 (no. 2382), phospho-IRS-1 (Ser307) (no. 2381), Akt (no. 9272), phospho-Akt (Ser473) (no. 9271), phospho-IKKα/β (Ser176/180) (no. 2694), SAPK/JNK (no. 9252), phospho-SAPK/JNK (Thr183/Tyr185) (81E11), rabbit mAb (no. 4668), β-actin (13E5) Rabbit mAb (no. 4970), SOCS3 (no. 2923), and anti-rabbit HRP-linked antibody (no. 7074) from Cell Signaling Technology (Beverly, MA) and Anti-IKK beta antibody [Y466] (ab32135) from Abcam (Cambridge, UK). After washing with Tris-buffered saline containing 0.1% Tween-20, all membranes were incubated for 1 h at 4°C with secondary antibody conjugated with a horseradish peroxidase. The specific immunoreactive bands were detected by chemilumininescence (GE Healthcare, ECL Plus Western Blotting Detection System, RPN2132). Images were acquired by ChemiDoc XRS Imaging System (BioRad) and quantified using Image Lab 3.0 software.
Results are expressed as mean ± SE. According to the Shapiro–Wilk W-test, the data were normally distributed and the homogeneity was confirmed by the Levene test. Therefore, one-way ANOVA was used to examine the effects of training and OT protocols on the studied parameters. When one-way ANOVA indicated significance, the Bonferroni post hoc test was performed. All statistical analyses were two-sided, and the significance level was set at P < 0.05. Statistical analyses were performed using STATISTICA 8.0 computer software (StatSoft®, Tulsa, OK).
Figures 1A and 1B show that the percentage alterations for exhaustion velocity and time to exhaustion were higher for TR (37.1 ± 2.9 and 81.2 ± 13.5, respectively) compared with C (−5.4 ± 2.0 and −3.3 ± 1.6, respectively) and OTR (−26.0 ± 2.5 and −59.8 ± 4.8, respectively). For exhaustion velocity, the OTR presented lower alteration compared with C. The percentage alterations between the blood lactate concentrations measured before the incremental load test and the peak blood lactate concentrations and the exhaustion time (min) measured at the end of week 8 were higher in TR (188.8 ± 38.5 and 41.0 ± 2.3, respectively) compared with C (73.5 ± 26.0 and 22.5 ± 1.3, respectively) and OTR (82.4 ± 31.1 and 28.4 ± 1.5, respectively) (Figs. 1C and 1D).
Figure 2A shows that TR and OTR presented lower body weight (g) at the end of week 7 (37.0 ± 0.8 and 36.7 ± 0.6, respectively) and week 8 (37.3 ± 0.9 and 37.2 ± 0.7, respectively) compared with C (39.0 ± 1.0 and 40.9 ± 1.0, respectively). The TR and the OTR presented lower percentage alterations for body weight (14.2 ± 1.1 and 13.5 ± 1.3, respectively) and food intake (12.9 ± 4.4 and 16.1 ± 2.9, respectively) compared with C (20.7 ± 1.9 and 40.5 ± 8.0, respectively) (Figs. 2B and 2C). Figures 2D and 2E show that blood glucose responses after an intraperitoneal injection of insulin and the corresponding area under curve from 0 to 30 min did not present significant differences between the experimental groups.
Figures 3A and 3B show that the pIRbeta was increased by 1.6- and 2.3-fold for EDL and by 1.7- and 2.7-fold for soleus muscles in the TR compared with the C and OTR after insulin injection, respectively. On the other hand, the pIRbeta was diminished by 0.7-fold for EDL and by 0.6-fold for soleus muscles in the OTR compared with C after insulin injection. Independently from insulin stimulation, the pIRS-1 was diminished by 0.5-fold for soleus muscles in the TR compared with C (Fig. 3D). On the other hand, the pIRS-1 was increased by 1.6-fold for EDL, and by 1.5- and 3.1-fold for soleus muscles in the OTR compared with C and TR, respectively (Figs. 3C and 3D). The pAkt was diminished by 0.7- and 0.6-fold for EDL and by 0.4- and 0.3-fold for the soleus muscles in the OTR compared with C and TR after insulin injection, respectively (Figs. 3E and 3F). On the other hand, the pAkt was increased by 1.5-fold for soleus muscles in the TR compared with C after insulin injection (Fig. 3F).
The pIKKalpha/beta increased by 1.4-fold for EDL in the TR compared with C (Fig. 4A). In addition, the pIKKalpha/beta increased by 1.8- and 1.3-fold for EDL, and by 9.0-fold for soleus muscles in the OTR compared with C and TR (Figs. 4A and 4B). The pSAPK/JNK increased by 1.4-fold for EDL in the TR compared with C (Fig. 4C). In addition, the pSAPK/JNK increased by 2.1- and 1.5-fold for EDL and by 3.1- and 3.7-fold for soleus muscles in the OTR compared with C and TR (Figs. 4C and 4D). The SOCS3 protein levels diminished by 0.4-fold for EDL in the TR compared with C (Fig. 4E), whereas the SOCS3 protein levels for soleus muscles increased by 1.5-fold in the TR compared with C (Fig. 4F). The SOCS3 protein levels increased by 4.1- and 9.5-fold for EDL and 2.1- and 1.5-fold for soleus muscles in the OTR compared with C and TR, respectively (Figs. 4E and 4F).
The main findings of the present investigation are as follows: (a) independently from the fiber type specificity, the OT protocol based on EE sessions decreased the muscle protein contents of pIRbeta (Tyr1146) and pAkt (Ser473) and increased the muscle protein contents of pIRS-1 (Ser307); and (b) independently from the fiber type specificity, the OT protocol based on EE sessions up-modulated the muscle protein contents of pIKKalpha/beta (Ser176/180), pSAPK/JNK (Thr183/Tyr185), and SOCS3. Taken together, our results show that overtrained mice presented muscle insulin signaling transduction impairment with concomitant increase of inflammatory proteins.
The results about Swiss mice performance evaluations (i.e., exhaustion velocity and time to exhaustion) and metabolic parameters (i.e., body weight and food intake) were reproduced for the experimental groups as previously verified (36,37). Regarding the decrease of food intake observed in TR compared with C, it is known that the increase of physical activity by the access to a running wheel is able to modulate both food intake and body weight in rodent obesity models (21,26,41). Ebal et al. (12) verified that lean Wistar rats performing moderate exercise during 5 wk diminished their food intake in 11% compared with their controls. The authors concluded that the feedback loop between corticosterone and ghrelin level was responsible by this result. In fact, the activation of the hypothalamo-hypophyso-adrenal axis with an anorexigenic effect has been reported even in the wheel-running model rat (5,8), in particular the corticotropin-releasing factor expression increase and/or the neuropeptide Y (NPY) expression decrease (3). We did not measure these parameters in our study; however, our results may be associated with these mechanisms (3,5,8,12).
In the current investigation, we presented the percentage alteration between the blood lactate concentrations measured before the incremental load test and the peak blood lactate concentrations (i.e., the higher values measured after the incremental load test). Interestingly, the TR showed higher blood lactate production compared with C and OTR (Fig. 1C). Once blood lactate concentration is sensitive to changes in exercise intensity and duration (2), our results may be explained because the exhaustion time of TR mice was also higher compared with C and TR (Fig. 1D).
There is a consensus in the literature about the positive effects of physical exercise on insulin action in states of insulin resistance (17,18). In fact, our research group has demonstrated the acute and chronic physical exercise-induced molecular mechanisms responsible by the improvement of muscle insulin signaling pathway in aging/obesity-induced insulin resistance (7,31,34,35,39,40). In addition, it is important to point out that the endurance training is able to improve the muscle insulin signaling pathway even without a previous insulin resistance state installed (1,6,27). However, here we showed that the effects of aerobic protocol (13,36,37) on the insulin signaling proteins were dependent on the muscle fiber type specificity. The pIRbeta (Tyr1146) and the pAkt (Ser473) were higher, and the pIRS-1 (Ser307) was lower in the soleus muscle samples of TR compared with C. However, only the pIRbeta (Tyr1146) was higher in the EDL samples of TR compared with C.
The regulation of IRS-1 serine phosphorylation is directly related to the molecular mechanism responsible by the impairment of insulin signaling pathway (20,43). On the basis of the previous information, it is possible to consider that the lack of improvement of pAkt (Ser473) in EDL samples of TR compared with C occurred partly due to the lack of difference of IRS-1 serine phosphorylation (Ser307) between these groups. This latter result may be explained by the modulation of other proteins in this specific tissue. For example, we verified that TR presented higher contents of pIKKalpha/beta (Ser176/180) and pSAPK/JNK (Thr183/Tyr185) in EDL samples compared with C. In fact, there are several evidences showing that these inflammatory proteins are involved in the inhibition of the skeletal muscle insulin signaling pathway through the increase of IRS-1 serine phosphorylation (Ser307) (7,31,38,39,42). In addition, we also demonstrated that C presented lower levels of myostatin compared with TR in EDL samples (37). Consistent results have been showing an inverse relationship between myostatin and Akt phosphorylation in skeletal muscles (19,30,45).
In contrast to other studies that verified positive effects of acute or chronic sessions of EE on insulin resistance parameters such as the homeostasis model assessment (HOMA) of human subjects (11,33) and the pAkt (Ser473) of lean Zucker rats (24), we showed that chronic sessions of EE decreased the pIRbeta (Tyr1146) and pAkt (Ser473) and increased the pIRS-1 (Ser307), pIKKalpha/beta (Ser176/180), pSAPK/JNK (Thr183/Tyr185), and SOCS3 compared with C and TR. The present findings are in accordance with Del Aguila et al. (10), which showed a decrease of IRS-1 tyrosine phosphorylation, association between IRS-1 and PI3-k, Akt serine phosphorylation, and Akt activity 24 h after an acute bout of downhill running performed by human subjects. Although the authors used the high serum values of TNF-α to justify their results (10,25), we did not find significant differences of this parameter measured in OTR compared with C and TR mice (37).
However, the high protein contents of IL-6, TNF-α, and myostatin observed in the skeletal muscles of OTR mice (37) are related to the impairment of insulin signaling pathway (15,19,44). In addition, the higher protein contents of pIKKalpha/beta (Ser176/180) and pSAPK/JNK (Thr183/Tyr185) of OTR compared with C and TR play a pivotal role on the impairment of the skeletal muscle insulin signaling pathway because of the increase of serine phosphorylation IRS-1 (7,31,38,39,42). Recently, Jorgensen et al. (23) verified that mice with muscle-specific deletion of SOCS3 fed with a high-fat diet presented higher IRS1-associated P85 subunit of PI3-k and Akt phosphorylation (Ser473 and Tyr308) after insulin treatment compared with wild-type mice. Therefore, the inhibition of the skeletal muscle insulin signaling pathway observed in the OTR mice, mainly the decrease of pAkt (Ser473), may also be associated with their high protein levels of SOCS3.
Although the EDL of TR and the EDL and soleus muscles of OTR presented impairment of insulin signaling pathway transduction, we did not verify significant differences during the insulin tolerance test (ITT) for the experimental groups. It is possible to consider that other tissues such as untrained skeletal muscles, liver, adipocytes, cardiac muscle, and hypothalamus may not present the inhibition of the insulin signaling pathway and play an important role in the maintenance of glucose homeostasis. Previous studies from our laboratory verified that diet-induced obese Wistar rats secreted high levels of insulin to compensate the insulin resistance that keeps the glucose levels similar to the control animals (7,34,40). Thus, we can consider that this physiological mechanism of compensation is active in Swiss mice. In addition, ITT was performed 1 wk before the end of the experimental protocols. It is important to point out that the OTR mice had their daily training sessions doubled during week 8 (36). These facts may have influenced our ITT results.
In conclusion, our study demonstrated that the effects of the aerobic training protocol on the insulin signaling pathway and on inflammatory proteins were dependent from the muscle fiber type specificity. Independently from the muscle fiber type specificity, the currently used OT protocol based on EE sessions impaired the insulin signaling pathway with concomitant increases of IKK, SAPK/JNK, and SOCS3 protein levels.
The present work received financial support from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, process nos. 2012/23258-0 and 2010/08239-4).
The authors are grateful for the technical support provided by Mrs. Simone Sakagute Tavares.
The authors declare no conflict of interest in the present study.
The results of the present study do not constitute endorsement by the American College of Sports Medicine.
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