Animals and ethical considerations
Male Wistar rats (Rattus norvegicus albinus) were obtained from the Center for Animal Experimentation (CEA) of University Center of Herminio Ometto Foundation – FHO (Araras, Sao Paulo, Brazil). The rats, weighing between 200 and 250 g, were placed in cages containing four animals and kept under controlled conditions of temperature (23 ± 1 ºC), humidity, and illumination (12 h light/dark cycles), with free access to water and rodent feed (Nuvilab) during the entire experiment.
All the experimental procedures were performed according to the protocols of the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health. The study also complied with the standards of the Brazilian College of Animal Experimentation (COBEA), Brazilian legislation on the scientific use of animals [22], and the Ethics Committee on Animal Use (CEUA) of Herminio Ometto Foundation - FHO, protocol 083/2014.
Induction of diabetes and experimental groups
The male Wistar rats were administered alloxan monohydrate (Sigma-Aldrich) dissolved in citrate buffer, at a dose of 32 mg/Kg, intravenously (in the penile dorsal vein), after 12 h of fasting. After this procedure, the animals were placed in cages and were administered a solution of glucose (15%) for 24 h, in order to avoid complications of alloxanic hypoglycemia [23]. One week after induction, blood samples were collected from the tail veins of the animals and the levels of glycemia were determined using a portable glucose meter (Accu-Chek Active). Rats with glycemia between 200 and 600 mg/dL were included in the subsequent experiments.
Thirty days after confirming hyperglycemia, the animals were randomly separated into the following groups: untrained control (C); trained control protocol (T); untrained diabetic (D); trained diabetic protocol (TD) (n = 8 rats/group). About the animals not induced to diabetes, it were made the same protocol, but without aloxane in solution injected (Fig. 1).
Physical exercise protocol
The physical exercise protocol was performed in a swimming tank with controlled water temperature of 31 ± 1 °C. First, the animals were adapted to the water environment for 10 days. For that, the animals were placed in individual bays (1 rat per bay, 12 bays per tank), separated by transparent acrylic divisions, in tanks (100 × 80 × 80 cm) with a maximum 50 cm height of water. The periods used were 5 min on 1st day, 10 min on 2nd day, and 15 min on 3th day, using a low water level [16]. From 4th day onwards, the water level was raised, requiring the animals to swim for 10 min on 4th day and for 15 min on 5th day. On 6th and 7th days, a load of 3% of the body weight was added to each rat for 10 and 15 min, respectively. The purpose of the adaptation was to reduce animals stress, without causing physiological adaptations [24].
Afterwards, a minimum lactate (ML) test was used to determine the aerobic/anaerobic metabolic transition, with an initial load of 13% of the body weight (for induction of hyperlactacidemia) and training for 30 sec. After resting for 30 sec, the rats were submitted to swimming with a 13% load until exhaustion. After resting for 9 min, a blood sample (25 µL) was collected from the distal tail vein for determination the lactate concentration, and the animals started exercise with progressively higher loads [24].The load was initially 2.0% of the body mass and was increased by 0.5% every 5 min, until exhaustion. At each load change, a blood sample (25 µL) was collected for lactate determination. At the end of each exercise, the rats were dried with a towel and returned to their cages. The ML was determined using a second order polynomial fit to the data of lactate plotted against the workload [24].
After the ML test, the exercise groups were submitted to the swimming exercise in individual tanks containing water at 31 ± 1 ºC, on six days per week, during four consecutive weeks, with lead weights attached to the thorax. The exercise training protocol used in the present study was based in a reverse periodization model in which intensity is initially at its highest and volume at its lowest. This protocol was chosen because it has been associated with increasing muscular endurance in humans [25]. Besides, it has been demonstrated a mean gain of 15% in the performance of rodents that exercise for 4 weeks at ML intensity [26]. Therefore, the initial training load was equivalent to 115% of the individual ML and the time spent on exercise totalized 25 min/day. Thereafter, an overload decrease of 5% was applied each week up to the 4th week (110% of ML in the 2nd week, 105% of ML in the 3th week and; 100% of the ML in the 4th week), with an increase of 5 min in the total period of activity (30 min in the 2nd week, 35 min in the 3th week and 40 min in the 4th week).
Glycemia
The glycemia test at the end of each training week (n = 8 animals per group). Blood samples were collected from the tail and it was determined using reagent strips and a glucose meter (Abbott, Chicago, USA). The glucose response during glycemia the end of each training week was calculated by estimating the total area under the curve using the trapezoidal method [27].
Harvesting of biological tissues
The animals were euthanized intraperitoneally with a solution of ketamine hydrochloride (75 mg/Kg) and xylazine hydrochloride (25 mg/Kg), 24 h after the final exercise session. The heart was removed and immersed in 14 mM KCl solution, followed by washing in ice-cold 0.9% NaCl and sectioning. The muscles that performed the most work in the swimming training were the red gastrocnemius (RG; 35–62% type I, 30–51% type IIA, 1–8% type IIB), containing a predominance of mixed fibers (oxidative/glycolytic), the white gastrocnemius (WG; 0% type I, 0% type IIA, 92% type IIB), with predominantly type IIB (glycolytic) fibers, and the soleus muscle (84% type I, 7% type IIA, 0% type IIB), with predominantly oxidative fibers. These muscles were removed and separated for biochemical (skeletal and cardiac muscles) [28] and histomorphometrical analysis (cardiac muscle).
Biochemical analyses of redox status (TBARS, H 2 O 2 , –SH groups, SOD and Catalase)
The white and red portions of the gastrocnemius muscle, the soleus muscle, and part of the left ventricle were homogenized in specific buffer using a Polytron Model PT 10/35 homogenizer (Brinkmann Instruments, Westbury, NY, USA) [29].
As a biomarker of oxidative stress, lipid peroxidation was detected by determination of MDA production. Briefly, samples of the RG, WG, soleus, and cardiac muscles were homogenized (using the Polytron device) in phosphate buffer and centrifuged at 5,000 rpm for 10 min at 18 °C. TBARS quantification was performed by colorimetric MDA analysis, using a spectrophotometer at 535 nm [4, 29].
H2O2 was determined by fluorescence (Amplex UltraRed Reagent kit, Life Technologies Corporation, Grand Island, New York, USA) [28].
As a biomarker of antioxidants, the quantification of -SH groups employed a colorimetric method involving reaction of the sulfhydryl group with 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), followed by spectrophotometric measurement at 412 nm [29].
SOD was measured by the inhibition of adrenaline oxidation, which was adapted from a previous study. Plasma samples were homogenized in glycine buffer. Volumes of 5, 10 and 15 µl of sample were separated after homogenization and 5,0 ml of catalase (0.0024 mg/mL of distilled water), 175–185 mL of glycine buffer (0.75 g in 200 mL of distilled water at 32 ºC, pH 10.2) and 5,0 µl of adrenaline (60 mM in distilled water plus 15 mL/mL fuming HCl) were added. The readings were performed for 180 sec at 10 sec intervals and measured on ELISA reader at 480 nm. Values were expressed as unit of SOD per milligram of protein (U/mg protein) [30]. Catalase activity was determined based on the rate of H2O2 decomposition by the enzyme present in the sample using 10 mM H2O2 in potassium phosphate buffer, pH 7.0. The maximum H2O2 decomposition rate was measured in a spectrophotometer at 240 nm, and values were expressed as catalase units per milligram of protein [31].
Histomorphometry
A portion of the heart was fixed in 10% paraformaldehyde buffered at pH 7.4 for 24 h, followed by dehydration, embedding in paraffin, and cutting into 5,0 µm sections. The sections were stained with Ferric Hematoxylin (to determine the number of cardiomyocytes - n/104 µm2) and Picro Sirius Red (to determine the percentage area of collagen fibers - % in 104 µm2) in order to evaluate the effects of the diabetic condition and the training protocols. Histological sections were photographed in the LEICA®DM-2000 optical microscope with LEICA®DFC-300 FX camera connected to the computer with LAS®software - Leica Application Suite for image capture Measurements were made using the Sigma Scan Pro 5.0 software [32] in triplicate for each animal, with determination of the group means from the sections from each animal.
Statistical analysis
All the data were first submitted to the Shapiro-Wilk normality test, followed by multifactorial analysis of variance (ANOVA Two-way) and Tukey's post-hoc. The results were expressed as means ± standard error mean and p < 0.05 was considered statistically significant. The statistical analyses were performed using GraphPad Prism 6.0 software.