Influence of the Use of Testosterone Associated with Physical
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original article Influence of the use of testosterone associated with physical training on some hematologic and physical parameters in older rats with alloxan-induced diabetes Romeu Paulo Martins Silva1,2, Rodrigo Otávio dos Santos2, Nelson Eurípedes Matildes Junior2, Antônio Vicente Mundim3, Mario da Silva Garrote-Filho3, Pâmella Ferreira Rodrigues2, Nilson Penha-Silva3 ABSTRACT 1 Centro de Ciências da Saúde e Objective: This study investigated the possible blood changes in wistar rats elderly with and without Desporto, Universidade Federal do treatment with anabolic steroids submitted physical training. Materials and methods: Elderly rats Acre (UFAC), Rio Branco, AC, Brasil (32) were divided into four groups: normal (N), treated normal (NT), diabetic (D) and treated diabetic 2 Centro Universitário do Planalto de (DT). They were submitted to 20 sessions of swimming with overload (5% body weight), 40 min/ Araxá (Uniaraxá), Araxá, MG, Brasil 3 Instituto de Genética e Bioquímica, day for four weeks. The NT and DT groups received application of testosterone twice a week. At Universidade Federal de Uberlândia the end of the sessions, the animals were subjected to swimming until exhaustion and then killed (UFU), Uberlândia, MG, Brasil for removal of blood and visceral fat. We evaluated maximum swim time, weight of visceral fat, erythrogram, leukogram, lipidogram and serum levels of glucose, lactate, aspartate aminotransferase Correspondence to: and creatine kinase. The results were compared using one-way ANOVA followed by the post hoc Romeu Paulo Martins Silva Tukey test. Results: In elderly diabetic rats, the use of anabolic associated with physical training in Universidade Federal do Acre Instituto de Genética e Bioquímica older rats resulted in improvement in erythrogram, lipidogram and physical performance for high- BR 364, km 04 intensity aerobic exercise. However, it was related to changes in leukocyte count, probably associated 69920-900 – Rio Branco, AC, Brasil with inflammation.Conclusion: The combination of the use of testosterone with physical training, [email protected] followed by maximal effort test caused changes hematological and biochemical can be associated with improvement in physiological characteristics, with increase of the swimming time and decrease of visceral fat levels, improvement in aerobic metabolism of fatty acids and glucose in normal and Received on March/5/2015 Arch Endocrinol Metab. 2017;61(1):62-9 Accepted on Jan/26/2016 diabetic animals. Keywords DOI: 10.1590/2359-3997000000200 Anabolic steroid; aging; diabetes; physical training INTRODUCTION by the testes and the adrenal (3). Testosterone promotes increased mass (4) and muscle strength (5). iabetes mellitus is characterized by a deficiency There is evidence that treatment with these in the production or action of insulin, or both, D compounds can improve the resistance of skeletal resulting in increased blood glucose in untreated muscle against fatigue, which increases the tolerance of individuals (1). experimental animals to physical activity (6). Diabetes can be induced in experimental animals by The intense physical exercise can alter the chemical substances such as alloxan or streptozotocin. concentration of blood cells and increase the use of These substances cause irreversible lesions in the glycogen, resulting in glucose fall (7,8) and increased pancreatic cells producing insulin. This causes a large production of lactic acid. reduction in the production of insulin, which results in Testosterone can also assist in modulating the diabetes (2). body’s immune response (9), by changing the activity The deficiency of insulin causes loss of muscle mass of leukocytes and other cells of defense (10). due to exacerbation of the decline of proteolysis and Physical exercise causes numerous physiological and protein anabolism. metabolic adjustments in the immediate or long term AE&M all rights reserved. © Besides insulin, protein anabolism is also stimulated so that the body can supply a higher energy demand Copyright by testosterone, androgen steroid hormone produced and to remain in homeostasis (11). 62 Arch Endocrinol Metab. 2017;61/1 Testosterone associated with physical training in older diabetes rats Although it is known that anabolic steroids can subjected to physical exercise with initial duration of 10 increase the tolerance to physical activity (6), the effects min. Every three days the duration of exercise increased of the combination of many synthetic anabolic steroids by 10 min until reach 40 min at the end of two weeks, with the exercise still need to be better known (12). with rest on Saturdays and Sundays. The purpose of this study was to analyze the effects After the adjustment period, the animals maintained of anabolic steroids on physical, hematological and the 40 min of physical exercise for six weeks, Monday biochemical variables in diabetic rats under aerobic to Friday, between 2 and 5 PM. This period of training physical training. was higher than achieved in other studies. The exercises consisted of sessions of swimming with moderate MATERIALS AND METHODS intensity, with 5% body weight tied to the tail. The animals swam in an adapted tank with depth of 48 cm and water The study was conducted in accordance with the temperature maintained between 30 and 36 °C (14). recommendations of the Brazilian College of Animal Experimentation (COBEA), with the National Council Treatment with anabolic for Animal Experiments Control (CONCEA) and with the Federal Law guidelines, 11,794, October 8, 2008 During the six weeks of physical training, the animals of – Brazil. the NT and DT groups received intramuscular injections of the mixture of esters of testosterone (testosterone Induction of diabetes by alloxan propionate, 30 mg; testosterone phenyl propionate, 60 mg; testosterone isocaproate, 60 mg; testosterone Induction of experimental diabetes mellitus followed decanoate, 100 mg; benzyl alcohol, 0.1 mL; and the protocols described in literature (13). After 24 hours of fasting, animals received intravenous injections (tail peanut oil, qs 1 mL) (Durateston™, Organon, Brazil), vein) of alloxan monohydrate (Sigma, St Louis, MO, with syringes of 1 mL (15 mg/kg body weight) twice a USA) in 0.01 M citrate buffer, pH 4.5 (35 mg/kg body week (on Tuesdays and Fridays at 4h30min PM). weight). The control rats underwent similar handling Animals not treated with the anabolic (groups N and but only with injection of a citrate buffer solution. D) received intramuscular injections of peanut oil (15). Two weeks after that treatment, rats that had levels of fasting glucose greater than or equal to 126 mg/dL were Test of maximum effort and sacrifice of animals considered diabetic and used in the study (1). At the end of six weeks of training, the animals were subjected to intense physical exercise until exhaustion. Experimental groups It was considered that the exhaustion was achieved when For this study we used 32 male albino rats (12 months) the animal could not keep their noses out of the water from Wistar race (Rattus norvegicus), distributed for more than 10 s. Once the exhaustion was reached, randomly into four groups (eight rats in each): normal the animals were removed from the water and placed animals without treatment (N), untreated diabetic on a bench. Body and tail were carefully dried with animals (D) normal animals with treatment (NT) and sterile paper towels. After the last session of exercise diabetic animals with treatment (DT). until exhaustion, the animals were anesthetized with ketamine and xylazine and sacrificed by decapitation Containment and nutrition in guillotine. Laparotomy was performed to collect The animals were kept in collective cages (100 x 50 x the retroperitoneal fat, mesenteric and epididymal in 30 cm) with an average of 3 rats per cage. The temperature analytical balance. inside the chamber of the vivarium was maintained between 22-25 °C in a room with controlled photoperiod Collection of blood samples set to 12 h of light and 12 h of darkness. All animals were The blood samples were taken at the end of six weeks of fed with standard balanced diet and water ad libitum. treatment. Before and after the exhaustion test, samples of 25 µl of blood were transferred from the tail of Adaptation and physical training each animal, using heparinized capillary glass, directly AE&M all rights reserved. The animals were subjected to an adjustment period into vials containing 50 µl of 1% sodium fluoride for © of two weeks. During this period, the animals were inhibition of glycolytic activity. These samples were Copyright Arch Endocrinol Metab. 2017;61/1 63 Testosterone associated with physical training in older diabetes rats used for determination of glucose and lactate. After the Statistical analysis exhaustion test, two blood aliquots were collected by The results between the different groups were compared cardiac puncture. The first sample (1 mL), collected in with use of ANOVA, with post hoc Tukey test, and were tubes containing K EDTA as anticoagulant, was used to 3 considered significantly different whenp < 0.05. The perform the hematologic analyses. The second sample tests were conducted using the application GraphPad (3 mL), collected in tubes without anticoagulant, was Prism 5 (GraphPad Software, San Diego, CA, USA). centrifuged at 720 x g and the serum was transferred to sealed vials and stored under refrigeration until the time of biochemical analysis. RESULTS Table 1 presents the average values obtained in the Dosage of glucose and lactate erythrogram of different groups. The concentration The blood glucose was determined using the Accu-Chek of hemoglobin, the red cell count and hematocrit apparatus (Roche, São Paulo, SP, Brazil). The blood values were higher in treated groups NT and DT in lactate was analyzed by electro-enzymatic method in relation to non-treated groups N and D (p < 0.05). But an automatic analyzer (YSI 1500 Sport L-Lactate, USI, there was no difference in these parameters between Yellow Springs, Ohio, USA). diabetic and normal groups in the absence and in presence of treatment with anabolic (p > 0.05).