Experiments were performed in male Wistar rats (100 – 120 g, ~30 days old) from the Animal House of the São Judas Tadeu University, São Paulo, Brazil. We chose male rats due to the high prevalence of MS and the elevated incidence of cardiovascular mortality in men . The animals were housed in collective polycarbonate cages in a temperature-controlled room (22°C) with a 12 h dark–light cycle (light 07:00 – 19:00 h). Rats were fed standard laboratory chow. The experimental protocol was approved by the Ethical Committee in Research of the Sao Judas Tadeu University (CEP - Protocol: 063/2006), and this investigation was conducted in accordance with the Principles of Laboratory Animal Care formulated by the National Institutes of Health (National Institutes of Health Publication No., 96–23, Revised 1996). The rats were randomly assigned into three groups: control (C, n = 8), sedentary fructose (F, n = 8), low intensity aerobic ET fructose (FT, n = 8).
Fructose drinking and exercise training
Fructose overload was performed via dilution of D-fructose in the drinking water (100 g/L) for a total period of 18 weeks. Control animals received only water during this period [9, 11]. Fructose consumption was measured daily, through the subtraction of the total volume given minus the remaining volume. The consumptions of chow and water (with or without fructose) were measured weekly. The total caloric intake was calculated considering that 2.89 kcal could be obtained from each consumed chow gram and that 4.0 kcal could be obtained from each ingested fructose gram .
During the tenth week of fructose overloading or water consumption, experimental groups were adapted to the treadmill (10 min/day; 0.3 km/h) and were submitted to a maximal treadmill exercise test. This exercise test was performed to determine aerobic capacity and ET intensity at the beginning of the exercise protocol (initial evaluation), after 4 weeks (to training intensity adjustments, data not show), and after ET protocol (final evaluation and aerobic capacity evaluation). Our group previously demonstrated that maximal treadmill exercise test can detect differences in aerobic performance, since that the maximum speed achieved in the test presented a good correlation with the maximum oxygen consumption . In the eleventh week of fructose overload, ET was started and performed on a motorized treadmill at low intensity (20-30% of maximal running speed) to FT group, for 1 hour a day, 5 days a week for 8 weeks [9, 11].
At the initial (initial evaluation) and after 8 weeks of ET protocol (final evaluation) the blood glucose and triglyceride concentrations were measured using a Roche device (Accutrend GCT, Roche, São Paulo, Brazil) after four hours of fasting. For the intravenous insulin tolerance test (ITT), the rats were fasted for two hours and then anesthetized with thiopental (40 mg/kg body weight, i.p.). A drop of blood was collected from the tail to measure the blood glucose concentration using the Accucheck system (Roche, São Paulo, Brazil) before and 4, 8, 12, and 16 minutes after insulin injection (0.75 U/kg, i.p.), as previously described by Bonora et al.  and published by our group [9, 11, 17]. The constant rate of decrease of the blood glucose concentration (Kitt) was calculated using the 0.693/t1/2 formula. The t1/2 for blood glucose was calculated from the slope of the least squares analysis of the blood glucose concentrations during the linear phase of decline [9, 11, 16, 17].
One day after final metabolic measurements, one catheter filled with 0.06 ml of saline were implanted in anesthetized rats (ketamine 80 mg/kg + xylazine 12 mg/kg, i.p.) into the carotid artery for direct measurements of the arterial pressure (AP), and into the jugular vein to vasoactive drugs administration (phenylephrine and sodium nitroprusside). One day after the catheter placement, the rats were conscious and allowed to move freely during the experiments. The arterial cannula was connected to a strain-gauge transducer (Blood Pressure XDCR, Kent© Scientific, Litchfield, CT, USA), and AP signals were recorded over a 30-min period by a microcomputer equipped with an analog-to-digital converter board (CODAS, 2-kHz sampling frequency, Dataq Instruments, Inc., Akron, OH, USA). The recorded data were analyzed on a beat-to-beat basis to quantify the changes in the mean AP (MAP) and the heart rate (HR) [9, 11, 17].
Sequential bolus injections (0.1 mL) of increasing doses of phenylephrine (0.25 - 32 mg/kg, i.v.) and sodium nitroprusside (0.05 - 1.6 mg/kg, i.v.) were given to induce increases or decreases in MAP responses (for each drug), ranging from 5 to 40 mm Hg. Baroreflex sensitivity was expressed as bradycardic response (BR) and tachycardic response (TR) in beats per minute per millimeter of mercury, as described elsewhere .
Systolic arterial pressure variability
Systolic arterial pressure (SAP, systograms) was obtained from blood pressure records. Fluctuations in SAP were further assessed in the frequency domain by means of autoregressive spectral estimation. The theoretical and analytical procedures for autoregressive modeling of oscillatory components have been described previously [18, 19]. Briefly, the SAP series derived from each recording were divided into 300 beat segments with a 50% overlap. The spectra of each segment were calculated via the Levinson-Durbin recursion and the order of the model chosen according to Akaike’s criterion, with the oscillatory components quantified in LF (0.2–0.6 Hz) and high-frequency (HF; 0.6–3.0 Hz) ranges [18, 19].
Tissue sample preparation
During the period of metabolic and hemodynamic evaluations (~1 week) the animals remained with fructose overload. Two days after hemodynamic measurements, the rats were killed. The animals were heparinized prior to fixation to optimize perfusion-fixation. White adipose tissue from different anatomical locations (perirenal, epididymal, mesenteric and subcutaneous) and the hearts (in diastole) were removed and weighed. The weight values of white adipose tissue presented in this study are the sum of the values taken from different anatomical locations. The myocardium was perfused through the aorta at a constant pressure of 80 mmHg using 0.1 M cacodylate buffer (3 min) followed by 2.5% glutaraldehyde solution diluted in cacodilate buffer. Posteriorly, the atria were separated from the ventricles, and the right from de left ventricle (LV) at the level of the papillary muscles, including the septum, was isolated. The ascending of aorta also excised and fixed with the same fixative solution for 24 h at temperature room.
Right atria and section left ventricle
Fragments of the right atria and section that included the entire thickness of the LV wall were divided into slices of approximately 3 mm wide and 5 mm long, fixed in 2% paraformadehyde, 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 2 h at 4°C and post-fixed in 1% osmium tetroxide in the same buffer for 2 h at 4°C. The fragments were dehydrated through a graded series of ethanol and embedded in Araldite. Thin sections were double-stained with uranyl acetate and lead citrate and examined with a JEOL – transmission electron microscope.
Fragments of the LV wall were fixed in formaldehyde solution 10%, buffered (pH 7.2) for 48 h, embedded in paraffin, and used for the histological slices, 6 μm thick, which were analyzed through polarized light microscopy with the use of Picrosirius staining in order to study the interstitial myocardial collagen fibers.
Ultrastructural morphometry and stereology
Ten electron micrographs from right atria per animal, chosen by systematic random sampling of squares, were taken at a final magnification of ×15 000 and the numerical density of granules/field, volume density of ANP-granules, mitochondrial and myofibrils and the diameter of all granules present in the field were determined, using a computerized program (Axio Vision, Zeiss). For the volume density the electronmicrographs were analyzed by a stereological test-system with 82 points, and values were expressed as a percentage.
Two randomly chosen blocks from each LV, in which the myocytes were cut in cross section, were used for quantitative analysis. The ultra-thin sections were placed on a copper grid, and 10 randomly chosen fields per block were selected for micrographs, which were taken from specimens using the Jeol transmission electron microscope. Low power (x600) electron micrographs were used for quantitative analysis of the LV muscle tissue composition. Each electron micrograph was analyzed by the computerized program (Axio Vision, Zeiss) totalling 300 micrographs. The myocyte mean cross-sectional area (A[my]) was determined for every animal in each group. A test-system with 140 sampling points was put upon the monitor screen and calibrated.
The myocardium was analyzed considering the myocytes (my), capillaries (cap) and connective tissue (ct). The numerical density (Nv) of cardiomyocytes (my per mm3) and capillaries (cap per mm3) was determined . The volume density was estimated for the myocyte (Vv [my]), capillaries (Vv[cap]) and collagens fibers (Vv[cf]): (Vv[structure] = PP[structure]/PT), where PP is the number of points that hit the structure, and PT is the total test-points. With the aid of the same test-system, the histological sections were used to estimate the volume density of the Picrosirius-stained collagen fibers was determined.
The ascending aorta was dissected (from heart base to the aorta arch), removed and post-fixed in 4% paraformaldehyde in 0.1 mol/l phosphate buffer, pH 7.2 for 24 h. Aortic rings were dehydrated in graded ethanol concentrations (70, 80, 90 and 100%) and embedded in histological paraffin. The blocks were cut with a microtome (5 – μm – thick sections, Leica). Transverse sections were mounted on a glass slide and stained with the Haematoxylin-Eosin, Verhoff-Van Gienson e Picrosirius technique. Four slides with 5 semi-serial (1 section every 25 μm) sections each, i.e. a total of 20 sections were obtained from each sample. Morphological analysis conducted in a transversal aortic sections with a light microscope (Zeiss, x200 and x400 magnifications) permitted the identification of elastic lamellas (Verhoff-Van Gienson stain), smooth muscle constituents (Haematoxylin stain) and collagen fibers (Picrosirius stain).
Ascendant aorta images were acquired and digitized for off-line morphometric analysis (Image Pro Plus 5.1 software). Four measures per image were obtained at 0, 90, 180 and 270° to estimate intima and media thickness (IMT). The aorta mean cross-sectional area (A[my]) was determined for every animal in each group. The lumen area (a) was estimated by drawing a line over the circle delimited by the intima layer inner interface. The lumen diameter (d) was calculated as: d = 2 √ a /π. The mean cross-sectional area of the intima plus media (IMA, intima-media area) was estimated as: IMA = [π(d/2 + IMT)2 − π(d/2)2]. IMA data were corrected for tissue shrinkage due to fixation and further processing by multiplying by 1.28 (previously determined in a pilot study).
Circumferential wall tension
Circumferential wall tension (CWT) was calculated by Laplace’s law as: CWT = MSAP × (d/2), where CWT is expressed in dyne/cm, MSAP is the mean of systolic arterial pressure (dynes/cm2), and d is the lumen diameter (mm) .
Images were captured with a light microscope (Zeiss, x400 magnifications), and transferred to the image analysis program (Axio Vision Software, Zeiss). For volume density of the Picrosirius-stained collagen fibers, the photomicrographs of the aorta were analyzed by a stereological test-system with 200 points, and values were expressed as a percentage.
Data are reported as mean ± SEM. After confirming that all continuous variables were normally distributed using the Kolmogorov-Smirnov test, statistical differences between the groups were obtained by 1-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls post-test. Statistical differences between the data measured over time were assessed using repeated measures ANOVA. Pearson’s correlation was used to study the association between different parameters. All tests were 2-sided, and the significance level was established at P < 0.05. Statistical calculations were performed using SPSS version 17.0.