The animals receiving the hypercaloric diet also had access to standard chow and water. The animals were weighed weekly, and the weight delta was defined as the difference between final and baseline weights. At the end of the experiment, the naso-anal length (cm) of the animals was measured to determine the Lee index. This index, which was adapted from Moura and Cols, corresponds to the ratio between the

cube root of the body weight (g) and the naso-anal length (cm) of the animals multiplied by 10 [21]. The liver, adrenal glands and specific adipose tissues (mesenteric, subcutaneous and visceral) were dissected manually and were weighed using a semi-analytical balance. The data were expressed as grams of tissue per 100 g of body weight (weight tissue/bodyweight × 100). The visceral adipose tissue weight included the perigonadal and retroperitoneal fat pads. The animals were Y-27632 solubility dmso killed by decapitation, and the blood and tissue samples were collected 24 h after the last session of restraint stress and after a 12-h fast. A trained practitioner performed the euthanasia. The trunk blood was collected and centrifuged for 5 min at 5000 × g at room temperature. This method was used to facilitate the collection of large volumes check details of blood serum for analysis. Importantly,

this model allows the determination of biochemical effects, including hormonal effects. The serum was frozen at −70 °C for subsequent analysis. The serum corticosterone levels were measured using a commercially available ELISA kit (Catalog No. 900-097, Assay Designs, Inc., USA), and the data are expressed as ng/mL. The serum leptin levels were measured using a commercial ever Linco ELISA Kit (Catalog No. 00EZRL-83, Linco Research, USA), and the data are expressed as ng/mL. The concentration of glucose, total cholesterol, HDL and TAG was measured spectrophotometrically using Bioliquid kits (Laborclin, Paraná, Brazil), and the data are expressed as mg/dL.

The VLDL and LDL values were calculated using the Friedewald equation (VLDL = TAG/5, LDL total cholesterol − (HDL–VLDL) [37]. The results were expressed as the mean ± standard error of the mean (S.E.M.). The baseline weight of the animals was compared between the groups using one-way ANOVA. The data and interactions were evaluated using two-way ANOVA (diet, stress, diet × stress) followed by Bonferroni correction for multiple comparisons when necessary and two-way ANOVA for repeated measures (effect of time, diet, stress, time × stress, time × diet, time × stress × diet, and diet × stress interactions) followed by Bonferroni correction when necessary. The between-group differences were considered significant at P < 0.05. The results of two-way ANOVA for repeated measures demonstrated an effect of time (F(5,30) = 77.863, P < 0.05) but no effect of stress (F(1,30) = 2.947, P > 0.05) or of hypercaloric diet (F(1,30) = 2.447, P > 0.05) ( Fig. 1, Panel A).