Effects of physical training on the immune system in diabetic rats

Int J Diab Dev Ctries | January-March 2010 | Volume 30 | Issue 1 Aims: This study aims to investigate the infl uence of physical training on the immune system of diabetic rats. Materials and Methods: Adult male Wistar rats were distributed into Sedentary Control (SC), Trained Control (TC), Sedentary Diabetic (SD) and Trained Diabetic (TD) groups were used. Diabetes was induced by alloxan (32 mg/bw-i.v.). Training protocol consisted of swimming, at 32 1 C, one hour/day, fi ve days/week, supporting an overload equivalent to 5% of the body weight, during four weeks. At the end of the experiment the rats were sacrifi ced by decapitation and blood samples were collected for glucose, insulin, albumin, hematocrit determinations, total and differential leukocyte counting. Additionally, liver samples for glycogen analyses were obtained. Results: The results were analyzed by one way at a signifi cance level of 5%. Diabetes reduced blood insulin, liver glycogen stores and increased blood glucose and neutrophil count. Physical training restored glycemia, liver glycogen levels, neutrophils and lymphocytes count in diabetic rats. Conclusions: In summary, physical training was able to improve metabolic and immunological aspects in the experimental diabetic rats.

The chronic insulinopenia and hyperglycemia of diabetes is associated with damage, dysfunction, and failure of various organs. [1]Regular exercise improves metabolic control in diabetic individuals and is an important component of treatment in diabetes mellitus.][7] On the other hand, studies reported that intense and/ or exhaustive training, long lasting acute exercises and insuffi cient terms of recuperation, lead to impairment of immune response.The decrease of serum glutamine has been cited as a factor that reduces the functionality of leucocytes increasing the susceptibility of athletes to infections.Skeletal muscles are the main source of serum glutamine, which is indispensable for leucocytes metabolism and functionality.ugiura and collaborators [8] studied the infl uence of chronic exercise training on immune system in voluntary running exercised rats.The protocol of voluntary running training was three days/week for eight weeks.Aft er the exercise program the rats showed improvement in macrophage and lymphocyte functions.

Introduction
Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action or both. [1]1] On the other hand, little is known about the role of moderate exercise on immune system of diabetic organisms.Therefore, in this study we investigated the eff ects of physical training on metabolism and leucocytes count in experimental diabetic rats.

Diabetes induction
Diabetes was induced by an intravenous injection (32 mg/kg b.w.) of Alloxan (Sigma).Aft er fi ve days, blood samples were obtained with animals in the fed state to determine the plasma glucose concentration.Rats which were not diabetic (Ͻ14,7 mmol/L) or too severely diabetic (Ͼ35,5 mmol/L) were eliminated from the study.

Training protocol
For the study, the rats were randomly distributed in four groups (n ϭ 8 per group), Sedentary Control (SC), Trained Control (TC), Sedentary Diabetic (SD) and Trained Diabetic (TD).The training included daily swimming with load of 5% of the body weight, one hour/ day, fi ve days/week, for four weeks.

Analyses
Hematocrit was measured at rest, to ensure that measurements of metabolite and hormone concentrations were not influenced by changes in plasma volume.A capillary tube sample of blood was spun at 3,000 revolutions/minute for three minutes and the hematocrit was determined.Then, the rats were sacrifi ced 48 hour aft er their last exercise bout and blood samples were collected for glucose, insulin, and albumin determinations, and diff erential and total leucocytes counting.Samples of liver were used to evaluate glycogen contents.
Serum glucose concentration was measured by a colorimetric method. [12]Serum insulin concentration was determined by radioimmunoassay (RIA, Kit Coat-A-Count, USA).Colorimetric methods were employed for the measurements of glycogen concentrations. [13]tal leucocytes count was performed using a specifi c pipett e and a Neubauer blood-counting chamber.For diff erential leucocytes counting, the leucocytes were stained with panoptic dye and observed on Zeiss microscope. [14]atistical analysis All dependent variables were analyzed by one-way analysis of variance (ANOVA) and a signifi cance level of P Ͻ 0.05 was used for all comparisons.The Bonferoni test was used for post-hoc comparisons.All results are expressed as mean Ϯ SD.

Results
Aft er the experimental period, alloxan-induced diabetes decreased serum insulin and liver glycogen.Diabetes also increased glycemia, whereas physical training recovered liver glycogen and decreased glycemia [Table 1].
Hematocrit and total leucocytes showed no diff erences among the experimental groups studied [Table 2].Table 3 shows that, in diff erential leucocytes, diabetes increased neutrophils.Neutrophils counts were recovered by

Discussion
The eff ects of exercise in diabetic organism are of great interest to scientifi c community.The benefi cial eff ects of physical activity on metabolic aspects are well known.Nevertheless, these effects on immune parameters remain uncertain.
As expected, alloxan induced-diabetes resulted in decreased blood insulin in both sedentary and trained rats.Diabetic animals showed hyperglycemia and the physical training decreased their blood glucose.[17] Several mechanisms may act locally to improve glucose uptake and disposal aft er exercise.These include increased muscle blood fl ow, increased insulin binding to its receptor (the insulin receptor, IR), increased IR turnover and increased glucose transport by stimulating GLUT4 translocation to the muscle cell surface. [15,18]Luciano et al. [19] showed that the increased responsiveness to insulin induced by chronic exercise in rat skeletal muscle may result, at least in part, from the modulation of the insulin signaling pathway at diff erent molecular levels.
The present study showed no diff erences in hematocrit among the groups, indicating that the alterations observed in blood parameters were not infl uenced by dehydration. [20]The study demonstrates that diabetes decreased liver glycogen content and the physical training counteracted this alteration.Many studies have demonstrated that liver glycogen is important to maintain physical performance during prolonged exercise. [21,22] The effects of insulin lead to a decrease in phosphorylase activity and an increase in glycogen synthase activity over that caused by glucose. [24]e exercise-induced adaptations observed in the present study are in agreement with other studies which showed that trained rats had a higher total hepatic glycogen synthetase activity a lower phosphorylase activity and increased glycogen contents in liver. [25]he effect of diabetes on the immune system has been the subject of much research. However, only a few studies att empted to elucidate the relationships among moderate physical training, diabetes mellitus and immune system.
Moderate exercise can decrease leucocyte adherence in blood vessels decreasing the incidence of chronic vascular diseases. [29]Leucocyte release during moderate endurance exercise is mainly related to demargination of cells adhering to the endothelium, this eff ect is caused by adrenergic stimulation and hemodynamic factors. [29]owever, McFarlin et al. [30] analyzed the affects of repeated bouts of moderate exercise in the same day on circulating leukocyte count.Total leucocyte count was increased aft er a bout of exercise and remained so aft er two hours.Nevertheless, aft er 24 hours, leucocyte count returned turned to basal levels.In the present study, sacrifi ce occurred 48 hours aft er the last exercise session, explaining the return to basal levels of leucocyte count.
A number of important changes in the expression of neutrophils adhesion have been described during and following exercise classifi ed in terms of the type, duration and intensity of the physical activity performed.Low intensities of endurance exercise do not aff ect either neutrophil adherence or the expression of adhesion molecules on neutrophils. [31]Perhaps the physical training protocol in our study did not interfere in neutrophil count because we utilized moderate intensity.
34] Neutrophils of diabetic patients do not increase their bactericidal activity in response to the same intensity of infection as compared to nondiabetic patients. [28]iabetes decreases the affi nity of the neutrophils with the endothelial cells.This eff ect, on the other hand, protects the lungs from neutrophils migration, thus decreasing its oxidative production.The increase in blood neutrophils observed might be a result of minor The increase in cardiac output resulted from adrenalin activity release which causes eosinophils movement and discharge from lung, spleen and liver.The reversal of this eff ect takes 24 hours.It is probable that we found no diff erences in response to exercise in the present study because the rats were sacrifi ced aft er 48 hours of the last training session. [33,34]sinophils are also altered in stress conditions.In the present study, the groups did not show interference from diabetes or physical training, indicating that the exercise swimming protocol here employed did not cause overtraining, since an increase in this parameter indicates overtraining. [35]me studies show an increase in the concentration of lymphocytes following exercise due to the recruitment of lymphocytes NK, cells T and B from the periphery of the body. [36]The present study showed that neither diabetes nor exercise caused alteration in the lymphocyte concentration.
Elevation of infl ammation markers is frequently reported in diabetic organisms with increase in subpopulations of monocytes, which may contribute to the development of atherosclerosis. [37]On the other hand, the exercise usually produced a decrease in some subpopulations of monocyte count. [38]Nevertheless, in our study neither diabetes nor exercise caused alteration in the monocyte percentage.

Conclusion
Our study showed that experimental diabetes induces metabolic damages and alters, at least in part, the diff erential leucocytes count.Also, moderate physical training was able to reverse the metabolic and immune parameters in diabetic rats.Future studies are required to evaluate the extent of the exercise infl uence in other experimental diabetes models.
Wistar rats were used in the experiments (180-210 g; 40-day-old).They were kept at 25ЊC with a light/dark cycle of 12 hour/12 hour, and fed with Purina rat food and water ad libitum.All experiments with the animals were performed in accordance with the specifi c Brazilian resolutions of the Bioethics of Experiments with animals (law NЊ 6.638 of May 8 th 1979; Decree NЊ 24.645 of July 10, 1934, Brazilian College of Animal Experimentation).

free from http://www.ijddc.com on Saturday, October 09, 2010, IP: 59.183.146.111] adherence
of cell in target tissues.Physical training was able to reverse this parameter as well and may have generated other positive adaptations to organism.