2.4.2. Plasma determinations
At the end of the treatment, mice were sacrificed under isoflurane anesthesia. Blood samples were collected in ice-cold tubes containing heparin and centrifuged at 5000 g at 4°C for 20 min, and the plasma was frozen at −80°C. Plasma glucose, low-density lipoprotein (LDL)-cholesterol, high-density lipoprotein (HDL)-cholesterol and triglycerides concentrations were measured by colorimetric methods using Spinreact kits (Spinreact, S.A., Girona, Spain). Plasma insulin concentrations were quantified using a mouse insulin ELISA kit (Alpco Diagnosis, Salem, NH, USA). Homeostatic model assessment of insulin resistance (HOMA-IR) was calculated using the formula: fasting glucose (mM)×fasting insulin (μU/mL)/22.5. Plasma levels of LPS were measured using a PierceTM Chromogenic Endotoxin Quant Kit (Thermo Scientific, Inc., Waltham, MA, USA) following manufacturer’s instructions. The plasma samples were pre-treated with a 10 mM MgCl2 solution and PyrosperseTMDispersing Agent (Lonza, Walkersville, MD, USA) in order to remove residual heparin used during blood extraction and fat, respectively. Sterile and pyrogen-free material was always used to guarantee sample and test integrity.