The animals fed with Standard Diet (Purina – Labina®) used for regular maintenance of our rats is composed of 50.30% of carbohydrate, 41.90% of protein and 7.80% of fat presenting a total of 2.18 kcal AZD6244 per 1 g of diet. High-fat diet was composed of 24.55% of carbohydrate, 14.47% of protein and 60.98% of fat, presenting a total of 5.28 kcal per 1 g of diet . The food intake was measured twice a week during the treatment to obtain food efficiency (food intake/body weight). Overnight fasted rats were killed by decapitation and samples of blood and epididymal, retroperitoneal
white adipose tissue and liver were collected, weighed and immediately frozen in dry ice and stored at −80 °C for subsequent analysis. For the glucose tolerance test, d-glucose (2 mg/g body weight) was intraperitoneally injected into overnight fasted rats. Glucose levels from tail blood samples were monitored at 0, 15, 30, 60, and 120 min. An insulin sensitivity test was performed on overnight-fed rats, after intraperitoneal injection of insulin (0.75 units/kg Sunitinib order body weight). Tail blood samples were taken at time
0, 15, 30, and 60 min after injection. Total serum cholesterol, high-density lipoprotein (HDL), triglycerides were assayed using enzymatic kits (Doles®, Goiania, Brazil). Enzyme-linked immunosorbent assay kits were used to measure serum adiponectin and insulin (Adipo-Gen®, Seoul, Korea) and leptin (Lincoln®, St. Louis, USA) levels. Total RNA from the liver was prepared using TRIzol reagent (Invitrogen Corp.®, San Diego, California, USA), treated with DNAse and reverse
transcribed with M-MLV (Invitrogen Corp.®) using random hexamer primers. The endogenous glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ACE, ACE2, resistin, TLR4, IL-6, TNF-α and NF-κB cDNA were amplified using specific primers and SYBR green reagent (Appllied Biosystems®, USA) in an PlusOne platform (Appllied Biosystems®). Relative comparative Ribonucleotide reductase CT method was applied to compare gene expression levels between groups, using the equation 2−ΔΔCT. Proteins were extracted from epididymal adipose tissue samples of rats and 30 μg of protein were resolved on SDS–PAGE gels (10%), transferred onto nitrocellulose membranes and blocked with Odyssey Blocking Buffer 1× (LI-COR Biosciences®, Germany). For immunoblotting, the membranes were probed with a polyclonal rabbit anti-p38/MAPK (Thr180/Tyr182) antibody (1:1000; Cell Signaling Inc., USA). The blots were then incubated with β-actin anti-rabbit IgG (1:1000; Sigma–Aldrich, Germany), was used as endogenous control. The blots were viewed using an infrared Q3127 LICOR® scanner and analyzed using the Odyssey® software.