•Excessive training leads to negative outcomes in multiple tissues.•Recent data about excessive training did not support the cytokine hypothesis.•Cytokines did not play a central role in performance ...decrement.
Chronic moderate-intensity exercise is an efficient non-pharmacological strategy to prevent and treat several diseases such as type 2 diabetes mellitus, cardiovascular and chronic obstructive pulmonary diseases, cancers, and Parkinson’s disease. On the other hand, improving an athlete’s performance requires completing high-intensity and volume exercise sessions. When the delicate balance between high-load exercise sessions and adequate recovery periods is disrupted, excessive training (known as overtraining) can lead to performance decline. The cytokine hypothesis considers that an imbalance involving excessive exercise and inadequate recovery induces musculoskeletal trauma, increasing the production and release of proinflammatory cytokines, mainly interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and interleukin 1beta (IL-1beta), which interact with different organic systems, initiating most of the signs and symptoms linked to performance decrement. This leading article used recent data to discuss the scientific basis of Smith’s cytokine theory and highlighted that the adverse effects of excessive exercise go beyond performance decline, proposing a multi-organ approach for this issue. These recent insights will allow coaches and exercise physiologists to develop strategies to avoid chronic excessive exercise-induced adverse outcomes.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The purpose of this study was to verify the effects of overtraining (OT) on insulin, inflammatory and gluconeogenesis signaling pathways in the livers of mice. Rodents were divided into control (CT), ...overtrained by downhill running (OTR/down), overtrained by uphill running (OTR/up) and overtrained by running without inclination (OTR). Rotarod, incremental load, exhaustive and grip force tests were used to evaluate performance. Thirty-six hours after a grip force test, the livers were extracted for subsequent protein analyses. The phosphorylation of insulin receptor beta (pIRbeta), glycogen synthase kinase 3 beta (pGSK3beta) and forkhead box O1 (pFoxo1) increased in OTR/down versus CT. pGSK3beta was higher in OTR/up versus CT, and pFoxo1 was higher in OTR/up and OTR versus CT. Phosphorylation of protein kinase B (pAkt) and insulin receptor substrate 1 (pIRS-1) were higher in OTR/up versus CT and OTR/down. The phosphorylation of IκB kinase alpha and beta (pIKKalpha/beta) was higher in all OT protocols versus CT, and the phosphorylation of stress-activated protein kinases/Jun amino-terminal kinases (pSAPK-JNK) was higher in OTR/down versus CT. Protein levels of peroxisome proliferator-activated receptor-gamma coactivator 1alpha (PGC-1alpha) and hepatocyte nuclear factor 4alpha (HNF-4alpha) were higher in OTR versus CT. In summary, OTR/down improved the major proteins of insulin signaling pathway but up-regulated TRB3, an Akt inhibitor, and its association with Akt.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Regular endurance exercise is a non‐pharmacological strategy to protect the liver against diseases. Conversely, exercise may be harmful when excessive, the so‐called overtraining. As expected, mice ...who underwent an overtraining protocol presented higher levels of proinflammatory cytokines in the serum and liver. Based on the relationship among overtraining, inflammation and mammalian target of rapamycin complex 1 (mTORC1) upregulation, the present study verified if animals submitted to an overtraining protocol, but with inhibition of the mTOR pathway via rapamycin injections could mitigate the liver and serum inflammation. Once autophagy can be linked to the improvement of hepatic dysfunction, we also investigated if the inhibition of mTORC1 by rapamycin can improve hepatic autophagy. The animals were randomized into four groups: control (CT; sedentary mice), overtraining by downhill running (OT; mice submitted to the downhill running‐based overtraining protocol), overtraining by downhill running with chronic administration of rapamycin (OT/Rapa; mice submitted to the downhill running‐based overtraining protocol with intraperitoneal injections of rapamycin) and aerobic (AER; submitted to aerobic training protocol). The serum and liver of the animals were used for biochemical analysis, reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR) and immunoblotting. The main results are (a) OT and OT/Rapa protocols decreased the performance; (b) the protein levels of interleukin 6 (IL‐6) were higher for the OT group; the OT/Rapa group reduced the autophagic genes, increased the microtubule‐associated protein light chain 3 II/I (LC3II/LC3I) protein ratio and decreased the sequestosome 1 (SQSTM1) protein. In conclusion, rapamycin appears efficiently to increase the autophagy proteins and decrease IL‐6 protein in the liver of overtraining mice.
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DOBA, FSPLJ, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
During overnight sleep, the longest postabsorptive and inactive phase of the day causes protein catabolism and loss. However, the daytime ingestion of dairy proteins has been shown to stimulate ...muscle protein synthesis and growth. This study compared the effects of pre-sleep supplementation of a protein blend (PB) composed of micellar casein (MCa) and whey protein (1:1) versus isolate MCa on the plasma levels of branched-chain amino acids (BCAAs) and the activation of the mechanistic target of rapamycin (mTOR) signaling, a critical intracellular pathway involved in the regulation of muscle protein synthesis. After 10 h of fasting during the active phase, rats were fed with a single dose of PB or MCa (5.6 g protein/kg of body mass) by gavage, and samples of blood and gastrocnemius muscle were collected at 30, 90, and 450 min. PB and MCa supplementations induced an increase (~3-fold, P < 0.001) of plasma BCAAs at 30 and 90 min. Most importantly, the stimulatory phosphorylation levels of mTOR and its downstream target p70 ribosomal protein S6 kinase (p70S6K) were similarly higher (~2.5-fold, P < 0.001) 30 and 90 min after MCa and PB. Plasma levels of leucine, isoleucine, valine, and overall BCAAs were correlated with the activation of mTOR (P < 0.001) and p70S6K (P < 0.001). MCa and PB supplementations before the inactive phase of rats resulted in an anabolic milieu in the skeletal muscle by inducing a transient increase in plasma BCAAs and a similar activation of the mTOR/p70S6K axis.
The transcriptional repressor REV-ERB-α, encoded by Nuclear Receptor Subfamily 1 Group D Member 1 (Nr1d1), has been considered to play an essential role in the skeletal muscle oxidative capacity ...adaptation and muscle mass control. Also, this molecule regulates autophagy via the repression of autophagy-related genes both in skeletal muscle and brain regions. Classically, training programs based on endurance or strength characteristics enhance skeletal muscle mass content and/or oxidative capacity, leading to autophagy activation in several tissues. Thus, it seems that REV-ERB-α regulates similar responses induced by exercise. However, how this molecule responds to different exercise models/intensities in different tissues is still unclear. Therefore, the main aim was to characterize the responses of REV-ERB-α and autophagy-related genes to different exercise protocols (endurance/interval run/strength) in distinct tissues (gastrocnemius, soleus and hippocampus). Since REV-ERB-α presents a circadian rhythm, the analyses were performed in a time-course manner. The endurance and strength groups attenuated REV-ERB-α transcriptional response during the time course in gastrocnemius and soleus. Conversely, the interval group enhanced the Nr1d1 expression in the hippocampus. All protocols downregulated the REV-ERB-α protein levels in gastrocnemius following the exercise session with concomitant nuclear exclusion. The major autophagy-related genes presented downregulation after the exercise session in all analyzed tissues. Altogether, these results highlight that REV-ERB-α is extremely sensitive to physical exercise stimuli, including different models and intensities in skeletal muscle and the hippocampus.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Chronic exercise induces cardiac remodeling that promotes left ventricular hypertrophy and cardiac functional improvement, which are mediated by the mammalian or the mechanistic target of rapamycin ...(mTOR) as well as by the androgen and glucocorticoid receptors (GRs). However, pathological conditions (i.e., chronic heart failure, hypertension, and aortic stenosis, etc.) also induce cardiac hypertrophy, but with detrimental function, high levels of proinflammatory cytokines and myostatin, elevated fibrosis, reduced adenosine monophosphate‐activated protein kinase (AMPK) activation, and fetal gene reactivation. Furthermore, recent studies have evidenced that excessive training induced an inflammatory status in the serum, muscle, hypothalamus, and liver, suggesting a pathological condition that could also be detrimental to cardiac tissue. Here, we verified the effects of three running overtraining (OT) models on the molecular parameters related to physiological and pathological cardiac hypertrophy. C57BL/6 mice performed three different OT protocols and were evaluated for molecular parameters related to physiological and pathological cardiac hypertrophy, including immunoblotting, reverse transcription polymerase chain reaction, histology, and immunohistochemistry analyses. In summary, the three OT protocols induced left ventricle (LV) hypertrophy with signs of cardiac fibrosis and negative morphological adaptations. These maladaptations were accompanied by reductions in AMPKalpha (Thr172) phosphorylation, androgen receptor, and GR expressions, as well as by an increase in interleukin‐6 expression. Specifically, the downhill running–based OT model reduced the content of some proteins related to the mTOR signaling pathway and upregulated the β‐isoform of myosin heavy‐chain gene expression, presenting signs of LV pathological hypertrophy development.
(a) Overtraining protocols induced left ventricle hypertrophy with signs of cardiac fibrosis and negative morphological adaptations. (b) The downhill running–based overtraining model reduced the content of some proteins related to the mechanistic target of the rapamycin signaling pathway and upregulated the β‐myosin heavy‐chain gene expression.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Although physical exercise-induced autophagy activation has been considered a therapeutic target to enhance tissue health and extend lifespan, the effects of different exercise models on autophagy in ...specific metabolic tissues are not completely understood. This descriptive investigation compared the acute effects of endurance (END), exhaustive (ET), strength (ST), and concurrent (CC) physical exercise protocols on markers of autophagy, genes, and proteins in the gastrocnemius muscle, heart, and liver of mice. The animals were euthanized immediately (0 h) and six hours (6 h) after the acute exercise for the measurement of glycogen levels, mRNA expression of
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, and protein levels of Beclin 1 and ATG5. The markers of autophagy were measured by quantifying the protein levels of LC3II and Sqstm1/p62 in response to three consecutive days of intraperitoneal injections of colchicine. In summary, for gastrocnemius muscle samples, the main alterations in mRNA expressions were observed after 6 h and for the ST group, and the markers of autophagy for the CC group were increased (i.e., LC3II and Sqstm1/p62). In the heart, the Beclin 1 and ATG5 levels were downregulated for the ET group. Regarding the markers of autophagy, the Sqstm1/p62 in the heart tissue was upregulated for the END and ST groups, highlighting the beneficial effects of these exercise models. The liver protein levels of ATG5 were downregulated for the ET group. After the colchicine treatment, the liver protein levels of Sqstm1/p62 were decreased for the END and ET groups compared to the CT, ST, and CC groups. These results could be related to diabetes and obesity development or liver dysfunction improvement, demanding further investigations.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Toll-like receptor 4 (Tlr4) is recognized due to its role in the immune response. Also, this protein can participate in the signaling pathway of events triggered by physical exercise such as ...apoptosis, inflammation, and endoplasmic reticulum (ER) stress. The main objective of this study was to evaluate the role of Tlr4 in the markers of these events in the myocardium of mice submitted to acute physical exercise (APE) protocols at different intensities.
Echocardiogram, RT-qPCR, and immunoblotting technique were used to evaluate the left ventricle of wild-type (WT) and Tlr4 knockout (Tlr4 KO) submitted to APE protocols at 45, 60, and 75% of their maximal velocity. Also, we performed the bioinformatics analysis to establish the connection of heart mRNA levels of Tlr4 with heart genes of inflammation and ER stress of several isogenic strains of BXD mice.
Under basal conditions, the Tlr4 deletion diminished the performance, and expression of inflammation and ER stress genes in the left ventricle, but increased the serum levels of CK, Il-17, and Tnf-alpha. Under the same exercise conditions, the Tlr4 deletion reduced the glycemia, serum levels of CK, Il-17, and Tnf-alpha, as well as genes and/or proteins related to apoptosis, inflammation and ER stress in the left ventricle, but increased the levels of CK-mb and LDH, as well as other genes related to apoptosis, inflammation, and ER stress in the left ventricle.
Altogether, the current findings highlighted the effects of different acute exercise intensities were attenuated in the heart of Tlr4 KO mice.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The protective effects of chronic moderate exercise-mediated autophagy include the prevention and treatment of several diseases and the extension of lifespan. In addition, physical exercise may ...impair cellular structures, requiring the action of the autophagy mechanism for clearance and renovation of damaged cellular components. For the first time, we investigated the adaptations on basal autophagy flux in vivo in mice's liver, heart, and skeletal muscle tissues submitted to four different chronic exercise models: endurance, resistance, concurrent, and overtraining. Measuring the autophagy flux in vivo is crucial to access the functionality of the autophagy pathway since changes in this pathway can occur in more than five steps. Moreover, the responses of metabolic, performance, and functional parameters, as well as genes and proteins related to the autophagy pathway, were addressed. In summary, the regular exercise models exhibited normal/enhanced adaptations with reduced autophagy-related proteins in all tissues. On the other hand, the overtrained group presented higher expression of
and
with negative morphological and physical performance adaptations for the liver and heart, respectively. The groups showed different adaptions in autophagy flux in skeletal muscle, suggesting the activation or inhibition of basal autophagy may not always be related to improvement or impairment of performance.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Interleukin 6 (IL-6) acts as a pro and anti-inflammatory cytokine, has an intense correlation with exercise intensity, and activates various pathways such as autophagy and mitochondrial unfolded ...protein response. Also, IL-6 is interconnected to circadian clock-related inflammation and can be suppressed by the nuclear receptor subfamily 1, group D, member 1 (
Nr1d1
, protein product REV-ERBα). Since IL-6 is linked to physical exercise-modulated metabolic pathways such as autophagy and mitochondrial metabolism, we investigated the relationship of IL-6 with REV-ERBα in the adaptations of these molecular pathways in response to acute intense physical exercise in skeletal muscle. The present study was divided into three experiments. In the first one, wild-type (WT) and IL-6 knockout (IL-6 KO) mice were divided into three groups: Basal time (Basal; sacrificed before the acute exercise), 1 hour (1hr post-Ex; sacrificed 1 hour after the acute exercise), and 3 hours (3hr post-Ex; sacrificed 3 hours after the acute exercise). In the second experiment, C2C12 cells received IL-6 physiological concentrations or REV-ERBα agonist, SR9009. In the last experiment, WT mice received SR9009 injections. After the protocols, the gastrocnemius muscle or the cells were collected for reverse transcription-quantitative polymerase chain reaction (RTq-PCR) and immunoblotting techniques. In summary, the downregulation of REV-ERBα, autophagic flux, and most mitochondrial genes was verified in the IL-6 KO mice independent of exercise. The WT and IL-6 KO treated with SR9009 showed an upregulation of autophagic genes. C2C12 cells receiving IL-6 did not modulate the
Nr1d1
mRNA levels but upregulated the expression of some mitochondrial genes. However, when treated with SR9009, IL-6 and mitochondrial gene expression were upregulated in C2C12 cells. The autophagic flux in C2C12 suggest the participation of REV-ERBα protein in the IL-6-induced autophagy. In conclusion, the present study verified that the adaptations required through physical exercise (increases in mitochondrial content and improvement of autophagy machinery) might be intermediated by an interaction between IL-6 and REVERBα.