Life Sciences 145 (2016) 144–151 Contents lists available at ScienceDirect Life Sciences j ourna l homepage: www.e lsev ie r .com/ locate / l i fesc ie Excessive eccentric exercise-induced overtraining model leads to endoplasmic reticulum stress in mice skeletal muscles Bruno C. Pereira a,1, Alisson L. da Rocha a,1, Ana P. Pinto a, José R. Pauli b, Claudio T. de Souza c, Dennys E. Cintra b, Eduardo R. Ropelle b, Ellen C. de Freitas d, Alessandro M. Zagatto e, Adelino S.R. da Silva a,d,⁎ a Postgraduate Program in Rehabilitation and Functional Performance, Ribeirão Preto Medical School, USP, Ribeirão Preto, São Paulo, Brazil b Sport Sciences Course, Faculty of Applied Sciences, State University of Campinas (UNICAMP), Limeira, São Paulo, Brazil c Exercise Biochemistry and Physiology Laboratory Postgraduate Program in Health Sciences, Health Sciences Unit, University of Far Southern Santa Catarina, Criciúma, Santa Catarina, Brazil d School of Physical Education and Sport of Ribeirão Preto, University of São Paulo (USP), Ribeirão Preto, São Paulo, Brazil e Laboratory of Physiology and Sport Performance (LAFIDE), Faculty of Sciences, Univ Estadual Paulista — UNESP, Bauru, São Paulo, Brazil ⁎ Corresponding author at: Avenida Bandeirantes, 39 Ribeirão Preto, São Paulo, Brazil. E-mail address: adelinosanchez@usp.br (A.S.R. da Silv 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.lfs.2015.12.037 0024-3205/© 2015 Elsevier Inc. All rights reserved. a b s t r a c t a r t i c l e i n f o Article history: Received 14 October 2015 Received in revised form 26 November 2015 Accepted 16 December 2015 Available online 18 December 2015 Aims: The present study verified the responses of selected endoplasmic reticulum (ER) stress proteins (i.e., BiP, ATF-6, pIRE1, pPERK, and peIF2alpha) in mice skeletal muscles after three different running overtraining (OT) protocols with same external load (i.e., intensity vs. volume), but performed in downhill, uphill and without in- clination. Materials and methods: The rodents were randomly divided into control (CT; sedentary mice), overtrained by downhill running (OTR/down), overtrained by uphill running (OTR/up) and overtrained by running without in- clination (OTR) groups. The incremental load test and exhaustive test were used as performance parameters. Forty hours after the exhaustive test performed at the end of the OT protocols (i.e., at the end of week 8) and after a 2-week total recovery period (i.e., at the end of week 10), the extensor digitorum longus (EDL) and soleus muscles were removed and used for immunoblotting. Key findings: For both skeletalmuscle types, the OTR/down protocol increased the pIRE-1, pPERK and peIF2alpha, which were not normalized after the total recovery period. At the end of week 8, the other two OT protocols up- regulated the BiP, pPERK and peIF2alpha levels only for the soleusmuscle. These ER stress proteinswere not nor- malized after the total recovery period for the OTR/up group. Significance: The above findings suggest that the OTR/down protocol-induced skeletal muscle ER stress may be linked to a pathological condition in EDL and soleus muscles. © 2015 Elsevier Inc. All rights reserved. Keywords: ER stress Mice Overtraining protocols Skeletal muscles 1. Introduction The endoplasmic reticulum (ER) is an intracellular organelle found in all eukaryotic cells and responsible for the biosynthesis, folding, as- sembly and modification of soluble and membrane proteins [17]. The ER also functions as a dynamic calcium storage responding to growth factors, hormones and stimuli that disrupt cellular energy homeostasis, nutrient availability or redox sate [36]. Physiological conditions increas- ing the protein folding demand or stimuli deregulating the reactions re- sponsible by the protein folding lead to an imbalance between the protein folding load and the ER capacity, which causes the accumulation of unfolded or misfolded proteins inside the ER lumen [35,36]. 00, Monte Alegre, 14040-900, a). This ER stress acts on the cells activating the unfolded protein re- sponse (UPR) to deal with stressful states and to solve protein folding defect [29,30]. The main UPR effectors are the activating transcription factor 6 (ATF6), the inositol requiring transmembrane kinase/endonu- clease 1 (IRE1) and the double-stranded RNA-dependent protein kinase (PKR)-like ER kinase (PERK). These effectors are released from the abundant ER chaperone immunoglobulin-heavy-chain-binding protein (BiP), activating the transcription of UPR target genes by the inhibition of the PERK-mediated eukaryotic translation-initiation factor 2alpha (eIF2alpha) [11], the autophosphorylation of IRE1 [29,30] and the mi- gration of a functional fragment of ATF6 to nucleus [12,34]. Considering the crosstalk between ER stress and inflammation, the IRE1 autophosphorylation alters its cytosolic domain allowing the bind of the adaptor protein tumor necrosis factor receptor-associated factor 2 (TRAF2), which activates the IκB kinase (IKK) and JUN N- terminal kinase (JNK), inducing the transcription of inflammatory genes [7,15,31]. The chronic increase of the interleukins 1-beta, 6 (IL- 1beta and IL-6) and tumor necrosis factor alpha (TNF-alpha) also http://crossmark.crossref.org/dialog/?doi=10.1016/j.lfs.2015.12.037&domain=pdf mailto:adelinosanchez@usp.br http://dx.doi.org/10.1016/j.lfs.2015.12.037 www.elsevier.com/locate/lifescie 145B.C. Pereira et al. / Life Sciences 145 (2016) 144–151 leads to ER stress, disrupting metabolic functions and causing more in- flammation [36]. The regular moderate-intensity exercise is used to prevent and treat several inflammatory processes [10,32] and the ER stress induced by this type of exercise acts as a protective mechanism against current and future stressors [8,18,33]. In contrast, studying a downhill running-based overtraining (OT) model, Pereira et al. [26,27] observed high levels of IL-6, TNF-alpha, IKK and JNK in serum and skeletal mus- cles of mice. According to Meeusen and coworkers [22], OT is defined as a process of intensified training that may lead to functional overreaching (FOR), nonfunctional overreaching (NFOR), or OT syn- drome (OTS). However, the responses of the ER stress in overtrained mice are unknown. Thus, we verified the effects of this OT protocol [26,27] on the BiP, ATF6, pIRE1 (Ser734), pPERK (Thr981) and peIF2alpha (Ser52) levels in mice skeletal muscles. Based on previous studies [7,15,31,36], we hypothesize that this OT protocol will up- regulate these proteins. Considering the singular characteristics of eccentric exercise [13] and knowing that other OT models were developed without the pre- dominance of this type of contraction [14], we also compared the re- sponses of the aforementioned ER stress proteins to the downhill running-based OT protocol [26,27] with other two OT protocols with same external load, but performed in uphill and without inclination [25]. Finally, to verify whether the effects of these OT protocols on the skeletal muscle ER stress are linked to pathological or non- pathological conditions [28], we measured the ER stress proteins after a 2-week total recovery period. 2. Methods 2.1. Experimental animals Eight-week-old male C57BL/6 mice were maintained in individual cages with controlled temperature (22 ± 2 °C) on a 12:12-h light– dark inverted cycle with food (Purina chow) and water ad libitum. The present work was approved by the Ethics Committee of the Univer- sity of Sao Paulo (ID 14.1.873.53.0) and adheres to the Brazilian law no. 11.794/2008 for the experimental use of animals. The rodentswere ran- domly divided into control (CT; sedentary mice; n = 12), overtrained by downhill running (OTR/down; performed the OT protocol based on downhill running; n=12), overtrained by uphill running (OTR/up; per- formed the OT protocol based on uphill running; n = 12) and overtrained by running without inclination (OTR; performed the OT protocol based on running without inclination; n = 12) groups. The mice were manipulated and/or overtrained in a dark room between 6 to 8 AM [24]. 2.2. Incremental load test (ILT) After adaptation to the treadmill running (INSIGHT®, Ribeirão Preto, São Paulo, Brazil) [24–27], the rodents performed the ILT. The initial Table 1 Responses of the incremental load test (m·min−1) and exhaustive test (s) to CT, OTR/down, O Incremental load test (m·min−1) Week 0 Week 8 Week 10 CT 24.5 ± 1.4 23.2 ± 1.3⁎ 22.7 ± 1.0 OTR/down 23.2 ± 1.0Φ 16.2 ± 1.0⁎,λ,Φ 15.5 ± 1.6 OTR/up 25.7 ± 0.8 18.8 ± 0.9⁎,λ 17.9 ± 1.0 OTR 24.4 ± 0.6 17.9 ± 1.8⁎,λ 16.1 ± 0.6 OTR/down: overtrained by downhill running; OTR/up: overtrained by uphill running; OTR: ov ⁎ P b 0.05 vs. week 0 for the same experimental group. # P b 0.05 vs. week 8 for the same experimental group. λ P b 0.05 vs. the CT group for the same experimental week. Φ P b 0.05 vs. the OTR/up group for the same experimental week. intensity of this test was 6 m·min−1 at 0% with increments of 3 m·min−1every 3 min until exhaustion, which was defined when each mouse touched the end of the treadmill 5 times in 1 min. The ro- dents were encouraged using physical prodding andwhen they became exhausted without completing the stage, the exhaustion velocity (EV; m·min−1) was corrected according to Kuipers et al. [20]. The EV of each mouse was used to prescribe the intensity of the OT protocols [24–27]. Onweek0, the experimental groups performed the ILTwithout inclination; however, at the end of weeks 4, 8, and 10, CT and OTR per- formed the ILT without inclination, OTR/down performed the ILT in downhill running, and OTR/up performed the ILT in uphill running [25]. 2.3. Running OT protocols, 2-week total recovery period and performance evaluations The 8-week running OT protocols performed in downhill, uphill and without inclinationwere applied as previously published [25], and each experimental week consisted of 5 days of training followed by 2 days of recovery. At the first four weeks of the OT protocols, the training inten- sity wasmaintained at 60% of the EV, the training volumewas gradually increased from 15min per day in the first week to 60min per day in the fourthweek, and rodents ran at a grade of 0%. At the fifthweek of theOT protocols, while the training intensity and volumeweremaintained, the rodents ran at a grade of−14% (i.e., OTR/down group), 14% (i.e., OTR/up group) and 0% (i.e., OTR group). At the sixth week of the OT protocols, the training intensity increased to 70% of the EV. At the seventh week of the OT protocols, the training intensity and volume increased to 75% of the EV and 75 min, respectively. At the eighth week of the OT protocols, the number of the training daily sessions increased from one to two with a rest interval of 4 h. The CT, OTR/down, OTR/up andOTR groupswere re-evaluated at the end of week 10. During these twoweeks (i.e., from the end of week 8 to the end of week 10), the rodents from OTR/down, OTR/up and OTR did not perform exercise sessions. The performance evaluations were ap- plied on week 0 and 48 h after the last sessions of the OT protocols at the end of weeks 8 and 10, and consisted of the ILT [24–27] and exhaus- tive test [24–27]. The exhaustive test was performed 24 h after the ILT and each mouse ran at 36 m·min−1 with 8% treadmill grade until ex- haustion, which was defined when the mice touched the end of tread- mill 5 times in 1 min. The rodents were encouraged using physical prodding. 2.4. Body weight and food intake The body weight and food intake of the experimental groups were registered daily. Indeed, the food intakewas determined by the subtrac- tion of the final food weight (i.e., the weight of the food put in the indi- vidual cage after 24 h) from the initial food weight (i.e., the weight of the food put in the individual cage on the previous 24 h) [25]. TR/up and OTR groups at weeks 0, 8 and 10. Exhaustive test (s) Week 0 Week 8 Week 10 ⁎ 59.0 ± 6.1 56.2 ± 10.0 57.6 ± 10.8 ⁎,#,λ 69.9 ± 6.5 16.5 ± 3.4⁎,λ 13.0 ± 2.1⁎,λ,Φ ⁎,λ 70.0 ± 6.8 17.4 ± 2.5⁎,λ 20.2 ± 3.0⁎,λ ⁎,λ 71.8 ± 6.1 17.1 ± 2.2⁎,λ 18.6 ± 3.8⁎,λ ertrained by running without inclination. Table 2 Responses of body weight (g) and food intake (g) to CT, OTR/down, OTR/up and OTR groups at weeks 0, 8 and 10. Body weight (g) Food intake (g) Week 0 Week 8 Week 10 Week 0 Week 8 Week 10 CT 22.7 ± 0.4 26.4 ± 0.4⁎ 26.8 ± 0.4⁎ 25.0 ± 1.2 26.3 ± 0.9⁎ 27.0 ± 0.8 OTR/down 21.0 ± 0.3λ 23.1 ± 0.5⁎,λ,Ϯ 24.6 ± 0.3⁎,λ,Φ 22.7 ± 0.8 22.1 ± 0.7λ,Φ,Ϯ 21.9 ± 0.9λ,Φ OTR/up 20.4 ± 0.4λ 24.1 ± 0.5⁎,λ 26.0 ± 0.4⁎,# 23.5 ± 0.8 28.8 ± 1.3⁎ 28.2 ± 1.4⁎,Ϯ OTR 21.3 ± 0.4λ 24.3 ± 0.2⁎,λ 25.6 ± 0.6⁎ 24.3 ± 0.5 24.6 ± 0.8Φ 21.7 ± 0.9#,λ OTR/down: overtrained by downhill running; OTR/up: overtrained by uphill running; OTR: overtrained by running without inclination. ⁎ P b 0.05 vs. week 0 for the same experimental group. # P b 0.05 vs. week 8 for the same experimental group. λ P b 0.05 vs. the CT group for the same experimental week. Φ P b 0.05 vs. the OTR/up group for the same experimental week. Ϯ P b 0.05 vs. the OTR group for the same experimental week. 146 B.C. Pereira et al. / Life Sciences 145 (2016) 144–151 2.5. Skeletal muscle extractions and immunoblotting analysis Themicewere anesthetized 40h after the exhaustive test performed at the end of the OT protocols (i.e., at the end of week 8) and after 2- week total recovery period (i.e., at the end of week 10). After a fast pe- riod of 6 h, themice were anesthetized with an intraperitoneal (i.p.) in- jection of 2-2-2 tribromoethanol 2.5% (10–20 μL·g−1). As soon as anesthesia was confirmed by the loss of the pedal reflexes, due to Fig. 1. Responses (arbitrary units) of BiP and its respective beta-actin (A), ATF-6 and its respe (Ser52)/eIF2alpha (E) measured at the end of week 8 in EDL for the experimental groups. Dat by downhill running; OTR/up: overtrained by uphill running; OTR: overtrained by running wit their different fiber type composition [2], the extensor digitorum longus (EDL) and soleusmuscles of both hindlimbswere removed and homog- enized in extraction buffer (1% Triton X-100, 100 mM Tris, pH 7.4, con- taining 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. ctive beta-actin (B), pIRE1 (Ser734)/IRE1 (C), pPERK (Thr981)/PERK (D) and peIF2alpha a correspond to means ± SE of n = 6 mice. CT: sedentary mice; OTR/down: overtrained hout inclination. *P b 0.05 vs. the CT group. **P b 0.05 vs. all experimental groups. Fig. 2. Responses (arbitrary units) of BiP and its respective beta-actin (A), ATF-6 and its respective beta-actin (B), pIRE1 (Ser734)/IRE1 (C), pPERK (Thr981)/PERK (D) and peIF2alpha (Ser52)/eIF2alpha (E) measured at the end of week 10 in EDL for the experimental groups. Data correspond to means ± SE of n = 6 mice. CT: sedentary mice; OTR/down: overtrained by downhill running; OTR/up: overtrained by uphill running; OTR: overtrained by running without inclination. *P b 0.05 vs. the CT group. **P b 0.05 vs. all experimental groups. †P b 0.05 vs. the OTR/down group. 147B.C. Pereira et al. / Life Sciences 145 (2016) 144–151 The extracts were centrifuged (9900 g) for 40min at 4 °C to remove the insolublematerial, and the supernatants of these homogenateswere used for protein quantification using the Bradford method [3]. 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 amount of protein employed for the immunoblotting analysis was 150 μg for both skeletal muscle samples. 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 sa- line (TBS) containing 5% BSA and 0.1% Tween-20, for 1 h, at 4 °C. The antibodies used for immunoblotting overnight at 4 °C were BiP (SC33757; 1:750), beta-actin (SC69879; 1: 750), PERK (SC13073; 1: 750), pPERK (Thr981; SC32577; 1: 750), eIF2alpha (SC11386; 1: 750) and peIF2alpha (Ser52; SC101670; 1: 750) from Santa Cruz Biotechnol- ogy (Santa Cruz, CA, USA); ATF-6 (NBP1-40,256; 1:1000) from Novus Biologicals (Littleton, CA, USA); IRE1 (AB37073; 1:1000) and pIRE1 (Ser724; AB104157; 1:1000) from Abcam (Cambridge, UK). After washed with TBS containing 0.1% tween-20, all membranes were incu- bated for 1 h at 4 °C with the appropriate secondary antibody conjugat- ed to horseradish peroxidase. The specific immunoreactive bands were detected by chemiluminescence (GE Healthcare, ECL Plus Western Blotting Detection System, RPN2132). The images were acquired by the C-DiGit™ Blot Scanner (LI-CORR, Lincoln, Nebraska, USA) and quan- tified using the software Image Studio for C-DiGit Blot Scanner. 2.6. Statistical analysis Results are expressed as themeans± standard error (SE). According to Shapiro–Wilk's W-test, the data were normally distributed and ho- mogeneity was confirmed by Levene's test. Therefore, the repeated- measures analysis of variance (ANOVA)was used to examine the effects of the experimental groups on the analyzed performance parameters, body weight and food intake. Planned comparisons were used to ana- lyze the present data. For each parameter, values within groups were compared between the experimentalweeks, in addition to comparisons of values between the groups in any given experimental week. The ef- fects of the experimental groups on the molecular parameters were evaluated by the ANOVA one-way. When repeated measures and/or one-way ANOVA indicated significance, Bonferroni's post hoc test was performed. All statistical analyses were two-sided and the significance level was set at P b 0.05. Statistical analyses were performed using STATISTICA 8.0 computer software (StatSoft®, Tulsa, OK). Fig. 3. Responses (arbitrary units) of BiP and its respective beta-actin (A), ATF-6 and its respective beta-actin (B), pIRE1 (Ser734)/IRE1 (C), pPERK (Thr981)/PERK (D) and peIF2alpha (Ser52)/eIF2alpha (E) measured at the end of week 8 in soleus for the experimental groups. Data correspond to means ± SE of n = 6 mice. CT: sedentary mice; OTR/down: overtrained by downhill running;OTR/up: overtrained by uphill running;OTR: overtrained by runningwithout inclination. *P b 0.05 vs. the CT group. **P b 0.05 vs. all experimental groups. ᵟP b 0.05 vs. the OTR/up group. 148 B.C. Pereira et al. / Life Sciences 145 (2016) 144–151 3. Results 3.1. Performance parameters, body weight and food intake Table 1 shows the responses of the incremental load test (m·min−1) and exhaustive test (s) to CT, OTR/down, OTR/up and OTR groups at weeks 0, 8 and 10. Considering the incremental load test, all the exper- imental groups decreased their performances at the end of weeks 8 and 10 compared to week 0. The OTR/down, OTR/up and OTR groups pre- sented lower performance levels compared to the CT group at the end of weeks 8 and 10. The OTR/down protocol decreased its performance at the end of week 10 compared to the end of week 8. The OTR/down group presented lower performance levels compared to the OTR/up group at the end of weeks 0 and 8. Considering the exhaustive test, the OTR/down, OTR/up and OTR groups presented lower performance levels at the end of weeks 8 and 10 compared to their own week 0, and to the CT group at the end of weeks 8 and 10. At the end of week 10, the OTR/down group presented lower performance levels compared to the OTR/up group. Table 2 shows that all experimental groups increased their body weighs at the end of weeks 8 and 10 compared to week 0. The OTR/ down, OTR/up and OTR groups presented lower body weights compared to the CT group at the end of weeks 0 and 8. At the end of week 8, the OTR/down group presented lower body weight compared to theOTR group. At the endofweek 10, theOTR/downgrouppresented lower bodyweight compared to the CT and OTR/up groups. The OTR/up protocol increased its body weight at the end of week 10 compared to the end of week 8. Regarding the food intake, the CT andOTR/up groups increased this metabolic parameter at the end of week 8 compared to week 0. At the end of week 8, the OTR/down group presented lower food intake compared to the other experimental groups, and the OTR group presented lower food intake compared to the OTR/up group. At the end of week 10, the OTR/down and OTR groups presented lower food intake compared to the CT group. The OTR/up protocol increased the food intake compared to its own week 0 and to the OTR/down and OTR groups at the end of week 10. Finally, the OTR protocol decreased this metabolic parameter compared to its own week 8. 3.2. EDL endoplasmic reticulum stress signaling At the end of week 8, the OTR/down protocol increased the BiP, ATF- 6, pIRE1 (Ser734) and peIF2alpha (Ser52) levels compared to the CT, OTR/up and OTR groups (Fig. 1A, B, C and E). Fig. 1A shows that the OTR/up protocol decreased the BiP levels compared to the CT group. Fig. 4. Responses (arbitrary units) of BiP and its respective beta-actin (A), ATF-6 and its respective beta-actin (B), pIRE1 (Ser734)/IRE1 (C), pPERK (Thr981)/PERK (D) and peIF2alpha (Ser52)/eIF2alpha (E) measured at the end of week 10 in soleus for the experimental groups. Data correspond to means± SE of n = 6mice. CT: sedentarymice; OTR/down: overtrained bydownhill running;OTR/up: overtrainedbyuphill running;OTR: overtrainedby runningwithout inclination. *Pb 0.05 vs. the CTgroup. **P b 0.05 vs. all experimental groups. λP b 0.05 vs. the OTR/down and OTR groups. 149B.C. Pereira et al. / Life Sciences 145 (2016) 144–151 The OTR/down and OTR/up protocols increased the pPERK (Thr981) levels compared to the CT group (Fig. 1D). After the 2-week total recov- ery period, Fig. 2A shows that the OTR/down and OTR/up protocols in- creased the BiP levels compared to the CT group. On the other hand, the OTR protocol decreased the BiP levels compared to the CT and OTR/down groups. Fig. 2B shows that the OTR protocol decreased the ATF-6 levels compared to the CT, OTR/down and OTR/up groups. The OTR/down protocol increased the pIRE1 (Ser734) and pPERK (Thr981) levels compared to all groups (Fig. 2C and 2D). While the OTR/down protocol increased the peIF2alpha (Ser52) levels compared to the CT and OTR groups, the OTR/up increased the phosphorylation of this pro- tein compared to all experimental groups (Fig. 2E). 3.3. Soleus endoplasmic reticulum stress signaling At the end of week 8, while the OTR/up protocol increased the BiP levels compared to the CT and OTR/down groups, the OTR protocol in- creased this protein expression compared to all experimental groups (Fig. 3A). Fig. 3B shows that the OTR/down protocol decreased the ATF-6 levels compared to all experimental groups. In addition, the OTR protocol decreased this protein expression compared to the CT and OTR/up groups (Fig. 3B). While the OTR/down protocol increased the pIRE1 (Ser734) levels compared to all experimental groups, the OTR/up protocol increased the phosphorylation of this protein com- pared to the CT group. In addition, the OTR protocol decreased the pIRE1 (Ser734) levels compared to the OTR/up group (Fig. 3C). Fig. 3D shows that the OTR/down, OTR/up and OTR protocols increased the pPERK (Thr981) levels compared to the CT group. In addition, the OTR/up protocol decreased the phosphorylation of this protein com- pared to the OTR/down and OTR groups. Fig. 3E shows that the OTR/ down, OTR/up and OTR protocols increased the peIF2alpha (Ser52) levels compared to the CT group, and the OTR/up protocol increased the phosphorylation of this protein compared to the OTR/down group. In addition, the OTR protocol increased the peIF2alpha (Ser52) levels compared to the OTR/down and OTR/up groups. After the 2-week total recovery period, the OTR/down protocol in- creased the BiP, pIRE1 (Ser734) and pPERK (Thr981) levels compared to all experimental groups (Fig. 4A, 4C and 4D). Fig. 4A shows that the OTR protocol decreased the BiP levels compared to the CT and OTR/up groups. The OTR/down and OTR protocols increased the ATF-6 levels compared to the CT and OTR/up groups (Fig. 4B). Fig. 4C shows that the OTR protocol decreased the pIRE1 (Ser734) levels compared to the CT and OTR/up groups. The OTR/up protocol increased the pPERK (Thr981) levels compared to the CT and OTR groups (Fig. 4D). While 150 B.C. Pereira et al. / Life Sciences 145 (2016) 144–151 the OTR/down protocol increased the peIF2alpha (Ser52) levels com- pared to the CT and OTR groups, the OTR/up protocol increased the phosphorylation of this protein compared to all groups (Fig. 4E). 4. Discussion The main findings of the present investigation are: a) In general, the OT protocols led to similar responses of the performance parameters as well as of the body weight and food intake; b) For both skeletal muscle types, the OTR/down protocol increased the most of the ER stress pro- teins, which were not normalized after the total recovery period; c) Only for soleus muscle, the other two OT protocols up-regulated the BiP, pPERK and peIF2alpha levels, which were partially normalized after the total recovery period. Taken together, the current results con- firmed our hypothesis showing that the OTR/down protocol is linked to an up-regulation of the skeletalmuscle ER stress proteins. Because these proteins were not normalized after the 2-week total recovery period, we suggest a possible pathological condition of ER stress in both skeletal muscle types. The responses of the exhaustion velocity and time to exhaustion were similar between the OT models measured at weeks 0, 8 and 10, and partially reproduced our recently published data [25]. The newest result of this study is that rodents from the different OT protocols did not improve their performance after the 2-week total recovery period. Because the performance repair occurs after atmost 14 days of recovery in the FOR state, we consider that these OT protocols induced the NFOR state, which is defined as a decrement or stagnation in performance that may be restored afterweeks ormonths of recovery andmaybe linked to psychological and hormonal disturbances [22]. Although this lack of performance repairwas previously observed for theOTR/downprotocol [24], herein we verified similar results for the OTR/up and OTR groups. The lower bodyweight of theOTgroups compared to the CT group at the end of week 8 may be related to the hypermetabolism and proteol- ysis under persistentworkloads [14,25] and reinforces that the decrease of this metabolic parameter is one of the classic symptoms of OT [1]. In- terestingly, after the recovery period, only the OTR/down group kept the low levels of bodyweight compared to the CT group. The food intake reduction is also considered a symptom of exhaustive training and OT [1,21]; however, only the OTR/down group was different from the CT group at the end of week 8. In contrast, the OTR/up group presented higher food intake compared to the other experimental groups, which reinforces that the uphill running demands more energy than the downhill running [4–6]. After the recovery period, while the OTR/ down and OTR/up groups kept the food intake levels observed at the end of week 8, the OTR protocol diminished this metabolic parameter. This last result corroborates Kaiyala and coworkers [16] showing that the acutely decreased energy expenditure diminishes food intake. Regarding the skeletal muscle ER stress, the OTR/down protocol up- regulated themost of the analyzed proteins in EDL and soleus at the end ofweek 8. Because the chronic increase of somepro-inflammatory cyto- kines induces ER stress [36], we consider these datamay be partially ex- plained by the high levels of IL-6 and TNF-alpha that were previously verified in both skeletal muscle types after the OTR/down protocol [27]. In addition, the current elevated levels of pIRE1 probably contrib- uted for the previously observed increase of JNK after the OTR/down protocol [26]. Wu and coworkers [33] verified skeletal muscle UPR acti- vation after one bout of exhaustive treadmill running; however, they showed that trained mice presented less activation or even repression of some proteins compared to untrained rodents after challenged with equal distance treadmill running. The authors concluded that the mod- erate training adapts UPR and protects skeletalmuscle from future exer- cise stress. In agreement, Kim and coworkers [19] verified that a 5-week high intensity training period decreased the ER stress in rat gastrocnemius muscle. According to Rayavarapu and coworkers [28], the moderate exercise-induced skeletal muscle ER stress is considered an adaptive mechanism that becomes pathological in situations in which an uncon- trolled ER stress leads to a crosstalkwith inflammatory activation. Based on the previously mentioned investigations [19,26–28,33], we suggest that the OTR/down protocol-induced skeletal muscle ER stress may be linked to a pathological condition. This hypothesis is reinforced by the high levels of most ER stress proteins for OTR/down group in EDL and soleus muscles even after the 2-week total recovery period. Except for the BiP and pPERK levels of the OTR/up group, the other ER stress proteins measured in EDL at the end of week 8 were not al- tered in response to OTR/up and OTR protocols. In order to justify these data, we consider two hypotheses. The first one reinforces the previously mentioned investigations [19,33] and suggests a positive ad- aptation of UPR in EDL after these OT protocols. Our second hypothesis is that themuscle recruitment pattern during running performed in up- hill andwithout inclinationmay be different compared to downhill run- ning. Therefore, further investigations should verify the electromyographic activity of EDL during these different OT protocols to test the preceding theory. Interestingly, after the recovery period, the OTR/up group displayed higher levels of peIF2alpha compared to the CT, OTR/down and OTR groups in EDL. Even a small phosphorylation of this protein in Ser51 drastically reduces the eukaryotic initiation factor 2B (eIF2B) activity [23], which actively participates in the protein synthesis of the skeletal muscle [9]. Regarding the soleus results at the end of week 8, the OTR/ up and OTR groups presented high levels of BiP, pPERK and peIF2alpha, which indicate UPR activation to deal with the exercise demands of these OT protocols. Even after the 2-week total recovery period, the OTR/up groupdisplayed high levels of pPERK andpeIF2alpha suggesting a possible pathological condition of ER stress for this skeletal muscle sample [28]. 5. Conclusion In summary, we confirmed our hypothesis showing that the OTR/ down protocol increased themost of the ER stress proteins in both skel- etal muscle types. Because most of these proteins were not normalized after the 2-week total recovery period, we suggest that the OTR/down protocol-induced skeletal muscle ER stressmay be linked to a patholog- ical condition. Even performed with the same external load, the effects of the OTR/up and OTR protocols on the ER stress proteins occurred ba- sically on soleusmuscle. Because the OTR/up group kept the high levels of pPERK and peIF2alpha after the total recovery period, we also consid- er a possible pathological condition of the ER stress in this specific skel- etal muscle type. Conflict of interest statement The authors declare no conflicts of interest. Acknowledgments The present work received financial support of São Paulo Research Foundation (FAPESP; process numbers 2013/19985-7, 2013/20591-3, 2014/25459-9 and 2015/08013-0). 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Introduction 2. Methods 2.1. Experimental animals 2.2. Incremental load test (ILT) 2.3. Running OT protocols, 2-week total recovery period and performance evaluations 2.4. Body weight and food intake 2.5. Skeletal muscle extractions and immunoblotting analysis 2.6. Statistical analysis 3. Results 3.1. Performance parameters, body weight and food intake 3.2. EDL endoplasmic reticulum stress signaling 3.3. Soleus endoplasmic reticulum stress signaling 4. Discussion 5. Conclusion Conflict of interest statement Acknowledgments References