Tnf α and il 6

All experimental procedures were performed in accordance with the Brazilian National Law and were approved by the by Ethics Committee of the Institute of Biomedical Sciences (ICB), University of São Paulo (USP) (number 195/11). Four-week-old C57BL/6 male mice were divided into two groups: (1) control group, which was fed a regular chow (70% carbohydrate, 20% protein, and 10% fat), with a caloric content of 3.8 kcal/g; (2) obese group, which was fed a high-fat diet (26% carbohydrate, 15% protein, and 59% fat) (PragSoluções, Brazil), with a caloric content of 5.4 kcal/g. The animals were maintained on a 12:12-hour light-dark cycle in a temperature-controlled environment (22 ± 2°C) with free access to food and tap water.
After 16 weeks subjected to a high-fat diet, the animals were fasted for 6 hours, weighed, and anesthetized with intraperitoneal injection of ketamine (150 mg/kg) and xylazine (7.5 mg/kg). Periepididymal and retroperitoneal fat deposits were excised and weighed. Blood samples were collected by cardiac puncture and the serum was used for biochemical assays. Triglycerides and total, HDL, and LDL cholesterol levels were measured by colorimetric kit assays (Labtest, Brazil). Glucose level was assessed by reactive strips (Johnson & Johnson, USA). Insulin (Cayman Chemical, USA) and TNF-α and IL-6 (BD Biosciences, USA) levels were assessed by ELISA assay kit.

Levels of IL-6 and TNF-α in the serum and tissue were measured using an enzyme-linked immunosorbent assay (ELISA), as previously described [22 (link)]. Briefly, 96-well plates (SPL Life Science, Seoul, Korea) were coated with 100 $\mu$ L of anti-mouse monoclonal antibody (1.0 mg/mL at pH 7.4 in phosphate buffered saline [PBS]) and incubated overnight at 4∘C. After additional washes, 50 μL of sample, or IL-6 and TNF-α (BD Bioscience, San Diego, CA, USA) standard was added and incubated at room temperature for 2 h. Plates were washed and 0.2 μg/mL of biotinylated anti-mouse antibody was added and incubated at room temperature for 2 h. After washing, avidin-peroxidase (Sigma-Aldrich, St. Louis, MO, USA) was added and plates were incubated for 30 min at 37∘C. The plates were then washed again and (2,2′-azino-bis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) substrate (BD Bioscience, San Diego, CA, USA) was added. Color development was measured at 405 nm using an automated microplate ELISA reader (Molecular Devices, Sunnyvale, CA, USA). Standard curves were prepared using serial dilutions of recombinant antibody (BD Bioscience, San Diego, CA, USA). Protein concentrations were measured using bicinchoninic acid (BCA) protein assay reagent (Sigma-Aldrich, St. Louis, MO, USA).