Adriana Cristina Levada-Pires

Effects of aerobic exercise training on antioxidant enzyme activities and mRNA levels in soleus muscle from young and aged rats

The aim of this study was to investigate the effect of aerobic exercise training on activities and mRNA levels of catalase (CAT), glutathione peroxidase (GPX), Cu,Zn- and Mn-superoxide dismutases (SOD), TBARS content, and xanthine oxidase (XO) activity, in soleus muscle from young and aged rats. The antioxidant enzyme activities and mRNA levels were markedly increased in soleus muscle with aging. TBARS content of soleus muscle from the aged group was 8.3-fold higher as compared with that of young rats. In young rats, exercise training induced an increase of all antioxidant enzyme activities, except for Cu,Zn-SOD. XO also did not change. The TBARS content was also increased (2.9-fold) due to exercise training in soleus muscle from young rats. In aged rats, the activities of CAT, GPX and Cu,Zn-SOD in the soleus muscle did not change with the exercise training, whereas the activities of Mn-SOD (40%) and XO (27%) were decreased. The mRNA levels of Mn-SOD and CAT were decreased by 42% and 24%, respectively, in the trained group. Exercise training induced a significant decrease of TBARS content (81%) in the soleus muscle from aged rats. These findings support the proposition that exercise training presents an antioxidant stress effect on skeletal muscle from both young and aged rats.

Palmitate increases superoxide production through mitochondrial electron transport chain and NADPH oxidase activity in skeletal muscle cells

The effect of unbound palmitic acid (PA) at plasma physiological concentration range on reactive oxygen species (ROS) production by cultured rat skeletal muscle cells was investigated. The participation of the main sites of ROS production was also examined. Production of ROS was evaluated by cytochrome c reduction and dihydroethidium oxidation assays. PA increased ROS production after 1 h incubation. A xanthine oxidase inhibitor did not change PA‐induced ROS production. However, the treatment with a mitochondrial uncoupler and mitochondrial complex III inhibitor decreased superoxide production induced by PA. The importance of mitochondria was also evaluated in 1 h incubated rat soleus and extensor digitorum longus (EDL) muscles. Soleus muscle, which has a greater number of mitochondria than EDL, showed a higher superoxide production induced by PA. These results indicate that mitochondrial electron transport chain is an important contributor for superoxide formation induced by PA in skeletal muscle. Results obtained with etomoxir and bromopalmitate treatment indicate that PA has to be oxidized to raise ROS production. A partial inhibition of superoxide formation induced by PA was observed by treatment with diphenylene iodonium, an inhibitor of NADPH oxidase. The participation of this enzyme complex was confirmed through an increase of p47phox phosphorylation after treatment with PA. J. Cell. Physiol. 216: 796–804, 2008,

Neurotoxicity of Anhydroecgonine Methyl Ester, a Crack Cocaine Pyrolysis Product

Smoking crack cocaine involves the inhalation of cocaine and its pyrolysis product, anhydroecgonine methyl ester (AEME). Although there is evidence that cocaine is neurotoxic, the neurotoxicity of AEME has never been evaluated. AEME seems to have cholinergic agonist properties in the cardiovascular system; however, there are no reports on its effects in the central nervous system. The aim of this study was to investigate the neurotoxicity of AEME and its possible cholinergic effects in rat primary hippocampal cell cultures that were exposed to different concentrations of AEME, cocaine, and a cocaine-AEME combination. We also evaluated the involvement of muscarinic cholinergic receptors in the neuronal death induced by these treatments using concomitant incubation of the cells with atropine. Neuronal injury was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. The results of the viability assays showed that AEME is a neurotoxic agent that has greater neurotoxic potential than cocaine after 24 and 48 h of exposure. We also showed that incubation for 48 h with a combination of both compounds in equipotent concentrations had an additive neurotoxic effect. Although both substances decreased cell viability in the MTT assay, only cocaine increased LDH release. Caspase-3 activity was increased after 3 and 6 h of incubation with 1mM cocaine and after 6 h of 0.1 and 1.0mM AEME exposure. Atropine prevented the AEME-induced neurotoxicity, which suggests that muscarinic cholinergic receptors are involved in AEMEs effects. In addition, binding experiments confirmed that AEME has an affinity for muscarinic cholinergic receptors. Nevertheless, atropine was not able to prevent the neurotoxicity produced by cocaine and the cocaine-AEME combination, suggesting that these treatments activated other neuronal death pathways. Our results suggest a higher risk for neurotoxicity after smoking crack cocaine than after cocaine use alone.

Neutrophil Death Induced by a Triathlon Competition in Elite Athletes

INTRODUCTION/PURPOSE The effect of a triathlon competition on death of neutrophils from elite athletes was investigated. METHODS Blood was collected from 11 sedentary volunteers and 12 triathletes under rest and after a Half Ironman triathlon competition (2-km swimming, 80-km cycling, and 20-km running). RESULTS The triathlon competition increased DNA fragmentation, phosphatidylserine externalization, and reactive oxygen species production in neutrophils when compared to the results at rest. The proportion of neutrophils with mitochondrial transmembrane depolarization was increased in the triathletes at rest and after competition as compared with sedentary volunteers. Plasma levels of thiobarbituric acid reactive substances were increased in triathletes after competition. Expression of bcl-xL (antiapoptotic) was decreased and that of bax (proapoptotic) was increased, whereas intracellular neutral lipid content was lowered in neutrophils after the triathlon. A positive correlation was found between the proportion of neutrophils with DNA fragmentation and the plasma free fatty acid levels (r = 0.688, P< 0.05), which was elevated by threefold after competition. Plasma levels of oleic, linoleic, and stearic acids were increased in triathletes after the competition when compared with sedentary volunteers. The plasma concentration of these three fatty acids, measured after the triathlon competition, was toxic for 3-h cultured neutrophils obtained from sedentary volunteers. The maximal tolerable (nontoxic) concentration of the fatty acids by 3-h cultured neutrophils was 100 micromol x L(-1) for oleic and linoleic acids and 200 micromol x L(-1) for stearic acid. CONCLUSION The triathlon competition induced neutrophil death possibly by apoptosis as indicated by DNA fragmentation and phosphatidylserine externalization. The increase in plasma levels of oleic, linoleic, and stearic acids induced by the competition may be involved in the neutrophil death observed possibly by increasing the production of reactive oxygen species and by decreasing the accumulation of intracellular neutral lipid.

The effect of glutamine supplementation and physical exercise on neutrophil function

Summary.Glutamine is the most abundant free amino acid in the body. Its primary source is skeletal muscle, from where it is released into the bloodstream and transported to a variety of tissues. Several studies have shown that glutamine is important for rat and human neutrophil function and that these cells utilize glutamine at high rates. Physical exercise has also been shown to induce considerable changes in neutrophil metabolism and function. As neutrophils represent 50–60% of the total circulating leukocyte pool and play a key role in inflammation, both physical exercise and glutamine might be expected to regulate the inflammatory process. In this review, the changes in neutrophil function induced by physical exercise and glutamine supplementation are compared.

Induction of Lymphocyte Death by Short- and Long-Duration Triathlon Competitions

PURPOSE The effect of triathlon competitions on death of lymphocytes from elite athletes was investigated. MATERIAL AND METHODS Blood was collected from sedentary volunteers and triathletes at rest and after a short-duration triathlon (SDT) and after a long-duration triathlon (LDT-half Ironman) competitions. RESULTS The athletes had lowered lymphocyte proliferation capacity compared with sedentary volunteers either at rest or after the competitions. There was no difference in the parameters associated with lymphocyte death when sedentary volunteers were compared with triathletes at rest. Lymphocytes from triathletes after SDT competition showed an increase in DNA fragmentation, phosphatidylserine externalization, and mitochondrial transmembrane depolarization and did not alter membrane integrity when compared with cells from athletes at rest. In contrast, the LDT competition raised the proportion of lymphocytes with loss of membrane integrity when compared with cells from athletes at rest and did not change the apoptotic parameters. The LDT competition induced an increase of reactive oxygen species (ROS) production by lymphocytes compared with triathletes at rest. The SDT competition did not alter ROS production by lymphocytes when compared with cells from triathletes at rest. ROS production by lymphocytes after LDT competition was 60% higher than in SDT. CONCLUSIONS Evidence is presented herein that an LDT competition caused lymphocyte death by necrosis, whereas an SDT induced lymphocyte apoptosis. The mechanism for lymphocyte death induced by the triathlon competitions may involve an increase in ROS production at different extents.

Glutamine Supplementation Stimulates Protein-Synthetic and Inhibits Protein-Degradative Signaling Pathways in Skeletal Muscle of Diabetic Rats

In this study, we investigated the effect of glutamine (Gln) supplementation on the signaling pathways regulating protein synthesis and protein degradation in the skeletal muscle of rats with streptozotocin (STZ)-induced diabetes. The expression levels of key regulatory proteins in the synthetic pathways (Akt, mTOR, GSK3 and 4E-BP1) and the degradation pathways (MuRF-1 and MAFbx) were determined using real-time PCR and Western blotting in four groups of male Wistar rats; 1) control, non-supplemented with glutamine; 2) control, supplemented with glutamine; 3) diabetic, non-supplemented with glutamine; and 4) diabetic, supplemented with glutamine. Diabetes was induced by the intravenous injection of 65 mg/kg bw STZ in citrate buffer (pH 4.2); the non-diabetic controls received only citrate buffer. After 48 hours, diabetes was confirmed in the STZ-treated animals by the determination of blood glucose levels above 200 mg/dL. Starting on that day, a solution of 1 g/kg bw Gln in phosphate buffered saline (PBS) was administered daily via gavage for 15 days to groups 2 and 4. Groups 1 and 3 received only PBS for the same duration. The rats were euthanized, and the soleus muscles were removed and homogenized in extraction buffer for the subsequent measurement of protein and mRNA levels. The results demonstrated a significant decrease in the muscle Gln content in the diabetic rats, and this level increased toward the control value in the diabetic rats receiving Gln. In addition, the diabetic rats exhibited a reduced mRNA expression of regulatory proteins in the protein synthesis pathway and increased expression of those associated with protein degradation. A reduction in the skeletal muscle mass in the diabetic rats was observed and was alleviated partially with Gln supplementation. The data suggest that glutamine supplementation is potentially useful for slowing the progression of muscle atrophy in patients with diabetes.

INFLAMMATORY RESPONSE AND NEUTROPHIL FUNCTIONS IN PLAYERS AFTER A FUTSAL MATCH

Abstract de Moura, NR, Cury-Boaventura, MF, Santos, VC, Levada-Pires, AC, Bortolon, JR, Fiamoncini, J, Pithon-Curi, TC, Curi, R, and Hatanaka, E. Inflammatory response and neutrophil functions in players after a futsal match. J Strength Cond Res 26(9): 2507–2514, 2012—Futsal players suffer injuries resulting from muscle fatigue and contact or collision among players. Muscle lesions can be detected by measuring muscle lesion markers such as creatine kinase (CK) and lactate dehydrogenase (LDH) in plasma. After an initial lesion, there is an increase in the plasma levels of C-reactive protein (CRP) and proinflammatory cytokines. These mediators may activate neutrophils and contribute to tissue damage and increase susceptibility to invasive microorganisms. In this study, we investigated the effect of a futsal match on muscle lesion markers, cytokines, and CRP in elite players. The basal and stimulated neutrophil responsiveness after a match was also evaluated based on measurements of neutrophil necrosis, apoptosis, phagocytic capacity, reactive oxygen species (ROS) production, and cytokines (tumor necrosis factor-alpha [TNF-&agr;], interleukin [IL]-8, IL-1&bgr;, IL-10, and IL-1ra) production. Blood samples were taken from 16 players (26.4 ± 3.2 years, 70.2 ± 6.9 kg, 59.7 ± 5.1 ml·kg−1·min−1, sports experience of 4.4 ± 0.9 years) before and immediately after a match. Exercise increased the serum activities of CK (2.5-fold) and LDH (1.3-fold). Playing futsal also increased the serum concentrations of IL-6 (1.6-fold) and CRP (1.6-fold). The TNF-&agr;, IL-1&bgr;, IL-8, IL-1ra, and IL-10 serum levels were not modified in the conditions studied. The futsal match induced neutrophil apoptosis, as indicated by phosphatidylserine externalization (6.0-fold). The exercise induced priming of neutrophils by increasing ROS (1.3-fold), TNF-&agr; (5.8-fold), and IL-1&bgr; (4.8-fold) released in nonstimulated cells. However, in the stimulated condition, the exercise decreased neutrophil function, diminishing the release of ROS by phorbol myristate acetate–stimulated neutrophils (1.5-fold), and the phagocytic capacity (1.6-fold). We concluded that playing futsal induces inflammation, primes and activates neutrophils, and reduces the efficiency of neutrophil phagocytosis immediately after a match.

Changes in lymphocyte and neutrophil function induced by a marathon race.

The aim of this study was to investigate the changes in lymphocyte and neutrophil selected functions before and after a marathon race. Fifteen professional athletes were recruited, and the following parameters were measured: plasma concentrations of IL‐1ra, IL‐6, IL‐8, IL‐10, TNF‐α and C‐reactive protein (CRP); neutrophil phagocytic capacity; cytokine production by neutrophils and lymphocytes and signs of neutrophil and lymphocyte death. The marathon race had no effect on CRP levels, but plasma concentrations of IL‐6 and IL‐1ra were increased. Although no effect was observed on the production of IL‐6, IL1‐ra, TNF‐α, IL‐1β and IL‐8 by unstimulated or stimulated neutrophils, a decrease in neutrophil phagocytic activity was observed immediately following the marathon. A high percentage of neutrophils undergoing apoptosis was observed due to the intense training regimen, whereas the percentages of apoptotic neutrophils were reduced after the race. The production of IL‐2, TNF‐α, IL‐1β and IL‐10 by lymphocytes was decreased by 50%–80%, and the percentage of apoptotic and necrotic lymphocytes was increased by 42% and fourfold, respectively, as a result of the race. In conclusion, the increase in plasma levels of IL‐6, IL‐8, IL‐1ra and IL‐10 after the race was not due to the production of the cytokines by neutrophils or lymphocytes. In fact, the marathon led to a decrease in lymphocyte and neutrophil function, and the diminished function was more pronounced in lymphocytes, indicating an impairment in acquired immunity. Copyright

Regulation of Gene Expression by Exercise-Related Micrornas

Gene expression control by microRNAs (miRs) is an important mechanism for maintenance of cellular homeostasis in physiological and pathological conditions as well as in response to different stimuli including nutritional factors and exercise. MiRs are involved in regulation of several processes such as growth and development, fuel metabolism, insulin secretion, immune function, miocardium remodeling, cell proliferation, differenciation, survival, and death. These molecules have also been proposed to be potential biomarkers and/or therapeutical targets in obesity, type 2 diabetes mellitus, cardiovascular diseases, metabolic syndrome, and cancer. MiRs are released by most cells and potentially act on intercellular communication to borderer or distant cells. Various studies have been performed to elucidate the involvement of miRs in exercise-induced effects. The aims of this review are: 1) to bring up the main advances for the comprehension of the mechanisms of action of miRs; 2) to present the main results on miR involvement in physical exercise; 3) to discuss the physiological effects of miRs modified by exercise. The state of the art and the perspectives on miRs associated with physical exercise will be presented. Thus, this review is important for updating recent advances and driving further strategies and studies on the exercise-related miR research.