Dianisidine

Trans-resveratrol, ethanol, dimethyl sulfoxide (DMSO), glucose oxidase, peroxidase, dianisidine and reagents used to determine fatty acids, triglycerides, lactate, reagents for Folch extraction, vanillin, KOH, amyloglucosidase, reagents used for preparation of buffer to liver lysis, reagents to Bradford method were provided from Sigma-Aldrich (St. Louis, MO, USA). Rabbit primary antibodies against insulin receptor were purchased from Santa Cruz Biotechnology (Dallas, TX, USA), and rabbit primary antibodies against pAMPK, pACC and β-actin were from Cell Signalling Technology, Inc. (Danvers, MA, USA) Polyvinylidene difluoride membranes were bought from Roche (Basel, Switzerland). SuperSignal® West Pico Chemiluminescent Substrate and CL-XPosure™ Film were purchased from Thermo Scientific. peroxidaseconjugated anti-rabbit antibody was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Kits for measurements of insulin, glucagon and adiponectin concentrations were from EMD Millipore (Billerica, MA, USA), and for γ-glutamyl transferase from Pointe Scientific (Canton, MI, USA).

Colon specimens were homogenized in 50 mM phosphate buffer (pH 6.0) and 0.5% hexadecyltrimethylammonium bromide (Sigma-Aldrich) using a gentleMACS tissue homogenizer (Miltenyi Biotech, Bergisch Gladbach, Germany). After three freeze and thaw cycles, supernatant was mixed with 0.02% dianisidine (Sigma-Aldrich) in 50 mM phosphate buffer, pH 6.0, and 0.0005% H2O2 (Sigma-Aldrich). Myeloperoxidase activity, expressed as arbitrary units, was calculated as mean absorbance (460 nm) per incubation time (in min) per protein content (in g).