Male Wistar rats (60-day-old) were randomly divided into two experimental groups to receive a control diet (CD, n=15) or a high-fat diet (HFD, n=15) for 30 weeks. The sample size and the experimental period were based on previous studies conducted by our research group.^9,11,12 Animals were kept in individual cages with controlled temperature (24 ± 2 ºC), humidity (55 ± 5%), and light (12-h light/dark cycle). The diet and water were ad libitum. The experimental procedures were performed according to the “Guide for the Care and Use of Laboratory Animals”^9,13 and approved by the Animal Ethics Committee of the Botucatu Medical School (991/2012). At the end of the experimental protocol, after 8h fasting, the animals were euthanized by decapitation after intraperitoneal anesthesia with a mixture of ketamine (1 mg/kg) and xylazine (100 mg/kg) (Syntec, Rhobifarma Indústria Farmacêutica Ltda., Hortolândia, São Paulo, Brazil). The blood and the cardiac samples were collected and stored at −20°C for further analysis.

The diets used in this study have been described elsewhere^11,12,14,15 and the composition followed AIN93 recommendations, consisting of the following ingredients: corn bran, soybean hull, soybean bran, dextrin, salt, vitamin and mineral complex, palm kernel oil, and soybean oil. The CD contained 31.0% of kcal from protein, 51.6% from carbohydrates, and 17.4% from fat. The HFD contained 18.7% of kcal from protein, 41.6% from carbohydrates, and 39.7% from fat. The content of saturated/unsaturated fatty acids was 61.5% / 38.5% in CD and 64.8% / 35.2% in HFD. The energetic densities from the diets were: HFD = 3.85 kcal/g and CD = 3.10 kcal/g.

The nutritional profile was determined by food and calorie intake, feed efficiency, final body weight, and adiposity index. Dietary intake and body weight were measured weekly. The calorie intake was determined by the following formula: weekly food intake multiplied by the energy value of each diet (g × kcal). To analyze the animal’s capacity to convert the consumed food energy in body weight, feed efficiency was calculated by dividing the total body weight gain (g) by the total energy intake (kcal). The total body fat was obtained by the sum of epididymal, retroperitoneal, and visceral deposits. The adiposity index was calculated by the total body fat divided by the final body weight and multiplied by 100.^15,16

A criterion based on the adiposity index was used to determine the occurrence of obesity and obesity resistance according to several authors.^10,17-19 After 30 weeks, the rats that consumed HFD were ranked based on their adiposity indexes. Therefore, the animals that received HFD and presented the highest adiposity indexes were classified as obese (Ob, n = 8); the animals that consumed a high-fat diet and presented adiposity indexes similar to control animals were classified as obesity-resistant (ROb, n = 6). Rats that failed to present the normal characteristic of the control group, while fed with a standard diet, were excluded (n = 8).

The metabolic evaluation included plasma lipid and glucose levels, as well as insulin resistance, whereas the hormonal evaluation was assessed by the concentrations of leptin and insulin.

The triglycerides, total cholesterol (TC), high- and low-density lipoprotein (HDL and LDL) levels were determined using a specific kit (BIOCLIN®, Belo Horizonte, MG, Brazil) and analyzed by the automated enzymatic colorimetric method (Chemistry Analyzer BS‐200, Mindray Medical International Limited, Shenzhen, China). The non-esterified fatty acid (NEFA) concentrations were evaluated by colorimetric kit (WAKO Pure Chemical Industries Ltd., Osaka, Japan).

Glycemia was analyzed in blood samples collected from the tails of the animals, using a handheld glucometer (Accu-Chek Go Kit, Roche Diagnostic Brazil Ltda, São Paulo, SP, Brazil). The homeostatic model assessment of insulin resistance (HOMA-IR) was used as an insulin resistance index, calculated according to the formula: HOMA-IR = [fasting glucose (mmol/L) x fasting insulin (µU/mL)]/22.5⁷.

The insulin and leptin levels were analyzed by the enzyme-linked immunosorbent assay (ELISA) method (EMD Millipore Corporation, Billerica, MA, USA). The reading was performed using a microplate reader (Spectra MAX 190, Molecular Devices, Sunnyvale, CA, USA).

The analysis was performed with live animals by transthoracic echocardiography, using a Vivid S6 system equipped with a multifrequency ultrasonic transducer 5.0 to 11.5 MHz (General Electric Medical Systems, Tirat Carmel, Israel). The animals were lightly anesthetized by intraperitoneal injection with a mixture of ketamine (50mg/kg) and xylazine (1mg/kg) and put in left decubitus position. Only one examiner performed all of the exams. The heart image structural measurements were obtained in one-dimensional mode (M-mode) guided by the images in two-dimensional mode with the transducer in the parasternal position, minor axis. Left ventricular (LV) evaluation was performed with the cursor M-mode just below the mitral valve plane at the level of the papillary muscles. The echocardiographic analysis was performed according to that established in prior studies.^19-21

The Systolic blood pressure (SBP) was measured by tail plethysmography, using a Narco Bio-System® Electro-Sphygmomanometer, model 709-0610 (International Biomedical, Austin, TX, USA). The animals were warmed in a wooden box (50 × 40 cm) between 38–40°C for 4–5 min to stimulate arterial vasodilation. After this procedure, a cuff with a sensor was placed in the proximal region of the tail, coupled to the electro‐sphygmomanometer. The cuff was inflated to 200 mmHg pressure and subsequently deflated.^15,16 The arterial pulsations were recorded in a computerized data acquisition system (AcqKnowledge ® MP100, Biopac Systems Inc., Santa Barbara, CA). The average of two readings was recorded for each measurement.

After euthanasia, the animals were submitted to thoracotomy, and the hearts, ventricles, and tibia were separated, dissected, weighed, and measured. Cardiac remodeling was determined by analyzing the weight of the heart and the left (LV) and right (RV) ventricles, and their correlation with the tibial length.

The Western Blot analysis was performed to evaluate the types I and III collagen protein expression. Briefly, the LV samples were rapidly frozen in liquid nitrogen and subsequently homogenized in a solution containing RIPA buffer (Amresco LLC, Solon, OH, USA), together with protease (Sigma-Aldrich, St. Louis, MO) and phosphatase (Roche Diagnostics, Indianapolis, IN, USA) inhibitors. The samples were subjected to SDS-PAGE in 10% polyacrylamide gel and were then electrotransferred to a nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ, USA). The blotted membrane was blocked (5% nonfat dry milk, 20 mmol/L Tris-HCl pH 7.4, 137 mmol/L NaCl and 1% Tween 20) for 2h at room temperature and then incubated overnight at 4–8°C with primary antibody against collagen type I (1:10000) and collagen type III (1:10000) (Abcam, Cambridge, MA, USA). The immunoblots were washed three times with TBS-T and incubated for 1.5h with peroxidase-conjugated anti-rabbit secondary antibody (1:2000) (Abcam, Cambridge, MA, USA), and then washed again three times with TBS-T and incubated with ECL (Enhanced Chemi-Luminescence, Amersham Biosciences, Piscataway, NJ, USA) for chemiluminescence detection. Blots were analyzed on Scion Image software (Scion Corporation, Frederick, MD, USA) and protein expressions were normalized to β-actin expression (1:1000) (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

The data were submitted to Kolmogorov-Smirnov normality test. Parametric variables were compared by One-Way analysis of variance (ANOVA) and complemented with the Tukey post hoc test for multiple comparisons when significant differences were found (p< 0.05). All the results are presented as mean ± standard deviation. Statistical analyses were performed using Sigma Stat for Windows Version 3.5. (Systat Software, Inc., San Jose, CA, USA). The level of significance considered was 5%.

Limitations of this study include the absence of histological analysis for collagen evaluation and the small sample size.