The effect of glutamine supplementation on the function of neutrophils from exercised rats

In a recent publication, we showed the protective effect of glutamine on neutrophil apoptosis induced by acute exercise. The purpose of the present study was to examine the effect of a single bout of intensive exercise on rat neutrophil function and the possible effect of glutamine supplementation. An aqueous solution of glutamine was given by gavage (1 g per kg b.w.), 1 h before the exercise session. The exercise was carried out on a treadmill for 1 h at 85% VO2máx.. Neutrophils were obtained by intraperitoneal lavage with PBS. The following parameters were evaluated: phagocytosis capacity, production of nitric oxide and reactive oxygen metabolites, expression of iNOS, and expression of NADPH-oxidase components (p22phox, p47phox and gp91phox). One hour of exercise at 85% VO2max. induced no change in the phagocytosis capacity and reactive oxygen species production but decreased nitric oxide production. When rats received oral glutamine supplementation, the phagocytosis capacity was significantly increased, the decrease in nitric oxide production induced by exercise was abolished and production of reactive oxygen species was raised. Glutamine supplementation presents a significant effect on neutrophil function including changes induced by exercise.     

Influence of a 3‐month training program on muscular damage and neutrophil function in male university freshman judoists

We studied the effects of a high intensity and high frequency 3‐month training program on muscle damage and neutrophil function in male judoists. The study included 15 male judoists who started intensive judo training program after a 6‐month break. Creatine kinase (CK), neutrophil counts and reactive oxygen species (ROS) production capability as well as phagocytic activity (PA) of neutrophils were measured at 2 stages; entering university (pre‐training) and after 3‐month training (post‐training). At both points, we investigated parameters three times: just before, immediately after and 24 h after a 2‐h practice session. Practice‐mediated change in CK was lower at post‐training than at pre‐training. Neutrophil count significantly increased after 2‐h practice but recovered 24 h later whereas it showed no subsequent and further increased at 24 h post‐practice. Although neutrophil ROS production capability and PA both decreased (breakdown) after practice session, ROS production capability increased and PA decreased (well‐adapted) at the post‐training. Long‐term training strengthened muscular function and improved neutrophil reaction against practice‐mediated stress. Copyright © 2012 John Wiley & Sons, Ltd.     

The influence of blood glucose on neutrophil function in individuals without diabetes

We assessed the association of neutrophil function with glycated hemoglobin (HbA1c) levels in a Japanese general population. Participants were 809 males and females who were over 20 years old living in the Iwaki region in Aomori Prefecture located in northern Japan. Lifestyle parameters (smoking, alcohol consumption, and exercise habits), HbA1c and neutrophil function such as reactive oxygen species (ROS) production capability and phagocytic activity (PA) were measured. ROS production capability was measured before and after phagocytic stimulus to obtain basal ROS production and stimulated ROS production. Level of HbA1c had a positive correlation with basal ROS production (p=0.053), a negative correlation with stimulated ROS production (p=0.072) and PA (p=0.059) only in post‐menopausal groups, and not in pre‐menopausal groups. However, there were no correlations between levels of HbA1c and neutrophil functions in male. In conclusion, in the present study, despite the presence of diabetes, chronic hyperglycemia was found to cause an increase in daily basal ROS production of neutrophils, and increased susceptibility to infection caused by reduced neutrophilic reaction in females in their menopause. Therefore, from the oxidative point of view, strict glycemic control is necessary to prevent post‐menopausal females from developing diabetic complications in spite of the presence of diabetes.     

The effect of glutamine supplementation and physical exercise on neutrophil function

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.     

Effects of supplementation with free glutamine and the dipeptide alanyl‐glutamine on parameters of muscle damage and inflammation in rats submitted to prolonged exercise

In this study, we investigated the effect of the supplementation with the dipeptide L ‐alanyl‐L ‐glutamine (DIP) and a solution containing L ‐glutamine and L ‐alanine on plasma levels markers of muscle damage and levels of pro‐inflammatory cytokines and glutamine metabolism in rats submitted to prolonged exercise. Rats were submitted to sessions of swim training for 6 weeks. Twenty‐one days prior to euthanasia, the animals were supplemented with DIP (n = 8) (1.5 g.kg^?1), a solution of free L ‐glutamine (1 g.kg^?1) and free L ‐alanine (0.61 g.kg^?1) (G&A, n = 8) or water (control (CON), n = 8). Animals were killed at rest before (R), after prolonged exercise (PE—2 h of exercise). Plasma concentrations of glutamine, glutamate, tumour necrosis factor‐α (TNF‐α), prostaglandin E2 (PGE2) and activity of creatine kinase (CK), lactate dehydrogenase (LDH) and muscle concentrations of glutamine and glutamate were measured. The concentrations of plasma TNF‐α, PGE2 and the activity of CK were lower in the G&A‐R and DIP‐R groups, compared to the CON‐R. Glutamine in plasma (p < 0.04) and soleus muscle (p < 0.001) was higher in the DIP‐R and G&A‐R groups relative to the CON‐R group. G&A‐PE and DIP‐PE groups exhibited lower concentrations of plasma PGE2 (p < 0.05) and TNF‐α (p < 0.05), and higher concentrations of glutamine and glutamate in soleus (p < 0.001) and gastrocnemius muscles (p < 0.05) relative to the CON‐PE group. We concluded that supplementation with free L ‐glutamine and the dipeptide LL ‐alanyl‐LL ‐glutamine represents an effective source of glutamine, which may attenuate inflammation biomarkers after periods of training and plasma levels of CK and the inflammatory response induced by prolonged exercise. Copyright © 2009 John Wiley & Sons, Ltd.     

Effects of dehydration on immune functions after a judo practice session

We investigated the effects of dehydration after a judo practice session on player muscle and immune functions. Subjects included 25 female university judoists. Investigations were performed before and after 2.5 h of regular judo practice. Body composition, serum enzymes (myogenic enzymes, immunoglobulins and complements), neutrophils counts, reactive oxygen species (ROS) production capability, and phagocytic activity (PA) were measured. Subjects were divided into two groups according to level of dehydration after practice (mild dehydration and severe dehydration groups) and results were compared. Creatine kinase was found to increase significantly after practice. In addition, neutrophil count also increased significantly after practice in both groups. The changing ratios of IgA, IgG and C3 observed in the mild dehydration group were significantly higher than those in the severe dehydration group. In the severe dehydration group, post‐practice PA/neutrophil had decreased significantly. Significant positive correlations were found between severity of dehydration and changing ratios of IgA, IgG, IgM, C3, C4 and ROS production capabilities, whereas no significant association was seen with PA and/or serum SOD activity. These results suggest that dehydration resulted in immunosuppression, including decreased neutrophil function. Copyright © 2012 John Wiley & Sons, Ltd.     

The effects of a two‐hour judo training session on the neutrophil immune functions in university judoists

The present study examined the effects of judo training on neutrophil and related functions. We measured and studied changes in the neutrophil and its related functions in 22 male university judoists immediately before (Pre values) and immediately after (Post values) a 2 h training session: reactive oxygen species (ROS) production capability, phagocytic activities (PA) and serum opsonic activity (SOA). Neutrophil count in whole blood, myogenic enzymes (creatine kinase, lactate dehydrogenase, aspartate aminotransferase and alanine aminotransferase), immunoglobulins (IgG, IgA and IgM) and complements (C3 and C4) in serum were also measured. The Post values of the neutrophil count, myogenic enzymes and IgG increased significantly compared with the Pre values. ROS production capability and SOA also significantly increased following training, although PA showed a slight decrease (but not statistically significant). Taking the findings of our previous studies into consideration, three major neutrophil or related functions, namely ROS production capability, PA and SOA, might compensate for each other to maintain the overall integrity of the neutrophil immune function, in that ROS and SOA increased to compensate for the slight decrease in PA, or PA slightly decreased to compensate for the increase in ROA and SOA after exercise. Copyright © 2008 John Wiley & Sons, Ltd.     

Granule localization of glutaminase in human neutrophils and the consequence of glutamine utilization for neutrophil activity

The provision of glutamine in vivo has been observed to reduce to normal levels the neutrophilia observed after exhaustive exercise and to decrease the neutrophil chemoattractant, interleukin-8. Thus, the role for glutamine in the regulation of inflammatory mediators of human neutrophil activation was investigated. The study sought to establish whether glutamine supplementation in vitro affects neutrophil function at rest and whether glutaminase, the major enzyme that metabolizes glutamine, is present in human polymorphonuclear neutrophils (PMN). During in vitro studies, the addition of 2 mm glutamine increased the respiratory burst of human PMN stimulated with both phorbol myristate acetate (PMA) and formyl-methionyl-leucyl-phenylalanine. These observations were made using a highly sensitive, real time chemiluminescent probe, Pholasin. Glutamine alone did not stimulate the release of reactive oxygen species. In a novel finding using glutaminase-specific antibodies in combination with flow cytometry and confocal microscopy, glutaminase was shown to be present on the surface of human PMN. Subcellular fractionation revealed that the enzyme was enriched in the secondary granules and could be released into cell culture medium upon stimulation with PMA. In conclusion, human PMN appeared to utilize glutamine and possess the appropriate glutaminase enzyme for metabolizing glutamine. This may depress some pro-inflammatory factors that occur during prolonged, exhaustive exercise.     

Competitive sustained exercise in humans, lymphokine activated killer cell activity, and glutamine – an intervention study

This study examined whether oral glutamine supplementation abolishes some of the exercise-induced changes in lymphocyte functions following long-term intense exercise. A group of 16 marathon runners participating in The Copenhagen Marathon 1996 were placed randomly in either a placebo (n = 7) or a glutamine receiving group (n = 9). Each subject received four doses of either placebo or glutamine (100 mg · kg⁻¹) administered at 0, 30, 60, and 90-min post-race. In the placebo group the plasma glutamine concentrations were lower than pre-race values during the post-exercise period [mean 647 (SEM 32) compared to 470 (SEM 22) μmol · l⁻¹ 90-min post-race, P < 0.05] whereas glutamine supplementation maintained the plasma glutamine concentration (at ∼750 μmol · l⁻¹). Glutamine supplementation in vivo had no effect on the lymphokine activated killer (LAK) cell activity, the proliferative responses or the exercise-induced changes in concentrations or percentages of any of the leucocyte subpopulations examined. Glutamine addition in in vitro studies enhanced the proliferative response in both groups. These data would suggest that decreased plasma glutamine concentrations post-exercise are not responsible for exercise-induced decrease in LAK activity and that the influence of glutamine in vitro is not dependent on the plasma glutamine concentration at the time of sampling. Accepted: 23 April 1998     

Changes in neutrophil functions during a 10‐month soccer season and their effects on the physical condition of professional Japanese soccer players

We examined changes in neutrophil function of soccer players over a 10‐month period and determined its effectiveness as an index for athlete physical condition. Subjects included 21 male professional Japanese soccer players. Data on body composition, myogenic enzymes and neutrophil function were obtained before and after 2 h of training at 3 investigation points: one week before opening season, at season mid‐point, and one week before the last game of the season. As a result, change ratios of myogenic enzyme levels before and after the 2‐hr training session at the third investigation point were significantly higher compared to the two other points. Reactive oxygen species production and phagocytic activity significantly increased after 2‐hr training session at point 1, although the extent of the increase became smaller over time and ROS production capability decreased significantly by point 3 assessment. Fatigue, especially muscle fatigue, chronically accumulated along with a gradual decrease in neutrophil immune function over the 10‐month season. Therefore, determination of neutrophil function can be used as a useful index to assess and understand an athlete's physical condition. Copyright © 2012 John Wiley & Sons, Ltd.     

Changes in immune functions during a peaking period in male university soccer players

The concept of peaking ensures that athletes have trained to attain their absolute peak performance levels prior to a competition. This study investigates the effects of peaking on the functions of neutrophils and lymphocytes in university soccer players during a five‐day soccer training camp followed by two weeks of tapering. The study subjects were 22 soccer players who were members of a university soccer club. We carried out our investigation during a five‐day training camp and two weeks after the training camp (i.e. the tapering period). We measured body composition, immune‐related parameters (leukocyte count, neutrophil count, lymphocyte count, immunoglobulins and complements), myogenic enzymes, superoxide dismutase activity (SOD) and neutrophil functions [reactive oxygen species (ROS) production, phagocytic activity, serum opsonic activity and lymphocyte subtypes]. Leukocyte and neutrophil counts tend to increase after the training camp compared with values before the training camp, and recovered during the conditioning period, although the final values were still lower than those before the training camp. The amount of ROS production per neutrophil and level of SOD decreased significantly during the conditioning period compared with before the training camp (p < 0.05, p < 0.01, respectively). Levels of Th1 cells decreased significantly during the conditioning period compared with the training camp (p < 0.05). In conclusion, the period of two weeks for the tapering period was considered insufficient to allow the athletes’ immune function to recover completely after an intensive training camp. Copyright © 2013 John Wiley & Sons, Ltd.     

The acute response of neutrophil function to a bout of judo training

Intensive exercise training decreases neutrophil functions in athletes. However, no studies to date have investigated the effect of irregular‐interval training, such as is associated with judo training programmes, on neutrophil functions. The purpose of this study was to examine such effects. Thirty‐seven male college judoists participated in this study. Neutrophil oxidative burst activity, phagocytic activity and expression of CD11b and CD16 per cell were measured by ?ow cytometry before and after judo training. Total neutrophil counts increased signi?cantly from 2.98 ± 0.82 to 7.95 ± 1.80 × 10³/µL (p < 0.001). The proportion of neutrophils producing reactive oxygen species (ROS) was increased signi?cantly (p < 0.001). On the other hand, the phagocytic activity decreased after training, as shown by a decrease in the amount of ingested opsonized zymosan per cell (p < 0.001), possibly as a compensatory effect for the increased numbers of ROS‐producing neutrophils. Expression of CD11b and CD16 per cell decreased by 20% and 30%, respectively, after judo training. In conclusion, judo training induced a decrease in phagocytic activity through the lowered expression of CD11b and CD16 on the surface of neutrophils, and increased the oxidative burst activity of neutrophils. Copyright © 2003 John Wiley & Sons, Ltd.     

Effects of 2.5‐hour sumo training on serum opsonic activity

Sumo is a traditional Japanese sport, but the effect of actual daily training on neutrophil function is unknown. We evaluated the effect of sumo training on serum opsonic activity (SOA), which is one of the main neutrophil‐related functions. Seventeen male university sumo wrestlers participated in the study. Changes in anthropometric parameters, concentrations of serum immunoglobulins (IgG, IgA and IgM), complements (C3 and C4), myogenic enzymes (lactate dehydrogenase, asparate aminotransferase, alanine aminotransferase and creatine kinase), white blood cell/neutrophil counts and SOA were measured immediately before and after actual daily training for 2.5 h. Compared with the pre‐values, immunoglobulins and complements, myogenic enzymes and white blood cell/neutrophil counts significantly increased (p <0.01 for all). As for SOA, the values of the peak height and the area under the curve significantly increased after the training when assessed using lucigenin as a chemiluminigenic probe (p <0.01 for all), but showed no significant change when luminol was used as the chemiluminigenic probe. In conclusion, daily actual sumo training for 2.5 h increases SOA, thus possibly activating the reactive oxygen species production of neutrophils. Copyright © 2009 John Wiley & Sons, Ltd.     

Effect of 6 months' training on the reactive oxygen species production capacity of neutrophils and serum opsonic activity in judoists.

The effects of long-term training on the production of reactive oxygen species (ROS) from neutrophils and serum opsonic activity (SOA) remain to date unknown. The aim of this study was to evaluate the effect of 6 months training on ROS production and SOA in judoists. Fifty-six judoists were enrolled this study. White blood cell counts, serum creatine kinase (CK), asparate aminotransferase (ASAT), alanine aminotransferase (ALAT), lactate dehydrogenase (LDH) and ROS production from neutrophils, and serum opsonic activity (SOA) using the lucigenin and luminol probes, were measured before and after daily judo exercise (2 h) in March and September. The subjects started their training from March after no exercise for three months, and continued it for 6 months (until September). In March, myogenic enzymes such as CK, ASAT, LDH and neutrophil counts increased and immunoglobulins, complements and SOA decreased after daily judo exercise. Such significant changes were not seen in September. On the other hand, ROS significantly increased after daily judo exercise in both March and September, with no significant difference in the rates of change. In conclusion, 6 month training minimized the changes in SOA as well as muscle enzymes, neutrophil counts, serum immunoglobulins and complements. This could be categorized as a long-term training effect. However, no such change was seen in ROS.     

Effects of soccer matches on neutrophil and lymphocyte functions in female university soccer players

In this study, changes in physical fatigue and biological functions of Japanese female soccer players were investigated by determining changes in neutrophil and lymphocyte functions. Study subjects included 18 female soccer players. Body composition, serum myogenic enzymes, neutrophil function, including reactive oxygen species (ROS) production capability, phagocytic activity (PA) and serum opsonic activity, as well as lymphocyte subpopulation were measured before and after a soccer match. Levels of myogenic enzymes (AST, ALT, CK and LDH) and immunoglobulins (IgG and IgA) and complements (C3) increased significantly after the match. In addition, leukocyte, neutrophils and lymphocyte counts increased whereas total PA decreased significantly. The number of T and Th1 cells (subsets of T helper cells) decreased whereas Th2 increased significantly. In addition, the number of B cells increased and NK cells decreased significantly after the match. The match was found to result in degenerative changes in and damage to athlete muscle tissues together with damage‐ and change‐mediated stress. These data also suggest a post‐match accelerated inflammatory reaction and potential immunosuppression as indicated by reductions in neutrophil PA and lymphocyte functions. Copyright © 2012 John Wiley & Sons, Ltd.     

The main neutrophil and neutrophil-related functions may compensate for each other following exercise-a finding from training in university judoists.

In order to clarify the relationship between exercise and neutrophil function, we measured three major neutrophil and neutrophil-related functions, viz. the reactive oxygen species (ROS) production capability and phagocytic activity (PA) of neutrophils and serum opsonic activity (SOA), simultaneously before and after a unified loading exercise under three different sets of conditions. Thirteen female collegiate judoists were examined with a unified exercise loading (2 h) immediately before and after a 64 day training period. Immediately thereafter, the athletes took part in a 6 day intensified training camp, following which the same exercise loading was repeated. Responses from circulating neutrophils were estimated by comparing the two sets of values obtained before and after the two instances of exercise loading. The parameters assessed included neutrophil count, SOA, PA and ROS production capability. ROS production increased after the exercise loading performed immediately before and after the 64 day training period just before the camp, (p < 0.01) but decreased following the exercise loading performed after the camp (p < 0.05). This suggested depressed bacteriocidal capability of the circulating neutrophils. PA decreased after the exercise loading sessions imposed prior to and after the 64 day training period (p < 0.01) but did not change in the loading session after the camp. No changes were seen in SOA produced with the loading exercise either before the 64 day exercise period or before the camp, but increased significantly following the post-camp session (p < 0.05). In conclusion, athletic training-induced changes in immune functional activities of neutrophils, such as ROS production and PA, and neutrophil-related factors, such as SOA, may compensate for each other to maintain the overall integrity of the neutrophil immune function.     

Glutamine supplementation further enhances exercise-induced plasma IL-6.

Exercise stimulates the production and release of interleukin-6 (IL-6) from skeletal muscle. Glutamine is also synthesized in skeletal muscle and is involved in protein synthesis within this tissue. During exercise, plasma levels of glutamine decline, and this may affect the concentration of plasma IL-6 via a decrease in IL-6 synthesis and release from muscle. We hypothesized that glutamine supplementation would attenuate the exercise-induced decrease in plasma glutamine concentration and, thus, further enhance levels of plasma IL-6. Eight healthy men participated in a randomized, double-blind, crossover study in which they performed 2 h of cycle ergometry at 75% of peak O2 uptake. They received glutamine, glutamine-rich protein, or placebo supplementation at intervals during and 2 h after exercise. Exercise induced an 11-fold increase in plasma IL-6, which was further enhanced by glutamine (18-fold) and glutamine-rich protein (14-fold) supplementation, administered at doses that attenuated the exercise-induced decrease in plasma glutamine concentration.     

Maternal voluntary exercise mitigates oxidative stress and incidence of congenital heart defects in pre‐gestational diabetes

Women with pre‐gestational diabetes have a higher risk of producing children with congenital heart defects (CHDs), caused predominantly by hyperglycemia‐induced oxidative stress. In this study, we evaluated if exercise during pregnancy could mitigate oxidative stress and reduce the incidence of CHDs in the offspring of diabetic mice. Female mice were treated with streptozotocin to induce pre‐gestational diabetes, then mated with healthy males to produce offspring. They were also given access to running wheels 1 week before mating and allowed to exercise voluntarily until E18.5. Heart morphology, gene expression, and oxidative stress were assessed in foetal hearts. Maternal voluntary exercise results in a significantly lower incidence of CHDs from 59.5% to 25%. Additionally, diabetes‐induced defects in coronary artery and capillary morphogenesis were also lower with exercise. Myocardial cell proliferation and epithelial‐mesenchymal transition at E12.5 was significantly lower with pre‐gestational diabetes which was mitigated with maternal exercise. Cardiac gene expression of Notch1, Snail1, Gata4 and Cyclin D1 was significantly higher in the embryos of diabetic mice that exercised compared to the non‐exercised group. Furthermore, maternal exercise produced lower reactive oxygen species (ROS) and oxidative stress in the foetal heart. In conclusion, maternal exercise mitigates ROS and oxidative damage in the foetal heart, and results in a lower incidence of CHDs in the offspring of pre‐gestational diabetes. Exercise may be an effective intervention to compliment clinical management and further minimize CHD risk in mothers with diabetes.     

Metabolic vulnerability of cisplatin‐resistant cancers

Cisplatin is the most widely used chemotherapeutic agent, and resistance of neoplastic cells against this cytoxicant poses a major problem in clinical oncology. Here, we explored potential metabolic vulnerabilities of cisplatin‐resistant non‐small human cell lung cancer and ovarian cancer cell lines. Cisplatin‐resistant clones were more sensitive to killing by nutrient deprivation in vitro and in vivo than their parental cisplatin‐sensitive controls. The susceptibility of cisplatin‐resistant cells to starvation could be explained by a particularly strong dependence on glutamine. Glutamine depletion was sufficient to restore cisplatin responses of initially cisplatin‐resistant clones, and glutamine supplementation rescued cisplatin‐resistant clones from starvation‐induced death. Mass spectrometric metabolomics and specific interventions on glutamine metabolism revealed that, in cisplatin‐resistant cells, glutamine is mostly required for nucleotide biosynthesis rather than for anaplerotic, bioenergetic or redox reactions. As a result, cisplatin‐resistant cancers became exquisitely sensitive to treatment with antimetabolites that target nucleoside metabolism.