Biochemical analysis in serum/plasma

Fasting levels of insulin and GLP-1 were assessed in plasma EDTA collected from the tip of the tail vein at baseline from either GTT or ITT assays in EDTA-coated tubes. Plasma was retrieved by centrifugation and stored at -20°C until assayed. Insulin and GLP-1 levels were evaluated by ELISA kits (Mouse Ultrasensitive Insulin ELISA, Mercodia AB, and Mouse Glucagon-Like Peptide 1 ELISA Kit, MyBioSource). All other circulating parameters were assessed on terminal serum collected through heart puncture at sacrifice. Serum was retrieved by centrifugation and stored at -20°C until assayed. LPS was measured by the Limulus Amebocyte Lysate assay (Pierce), and triglycerides by TGs Colorimetric Assay KIT (CAY-10010303-96, CaymanChem). Low-Density Lipoprotein Cholesterol (LDL-C) and High-Density Lipoprotein Cholesterol (HDL-C) were measured through colorimetric assay kits (E-BC-K205 and E-BC-K221, respectively from Elabscience), while total cholesterol was calculated crossing HDL-C, LDL-C and TGs values of each individual mouse (applying Friedewald’s formula). Alanine aminotransferase levels were measured through colorimetric tests (ALT color Microwell, Sentinel Diagnostics). All the analyses were performed following manufacturers’ instructions.