Nefa fs

The miR-320-3p has been associated with the regulation of Glucose-Induced Gene Expression in Diabetes (Feng and Chakrabarti, 2012 (link)). At sacrifice, rat pup was sampled of one blood drop to determine the total blood glucose by AccuChekH Active (Roche-Diagnostics GmbH, Mannheim, Germany), and blood plasma was collected on EDTA tubes (Wang et al., 2012 (link)), after centrifugation at 2,000 × g for 10 min and stored at −80°C. Cholesterol (Cholesterol FS^*, DiaSys Diagnostic Systems GmbH), Triglycerides (Triglycerides FS^*, DiaSys Diagnostic Systems GmbH), and Non-Essential Free fatty acids (NEFA FS^*, DiaSys Diagnostic Systems GmbH, Holzheim, Germany) were measured on rat plasma.

Plasma insulin and GLP-1 were measured with ELISA kit (STELLUX® Chemiluminescent Rodent Insulin ELISA ALPCO and EZGLP1T-36K, Millipore, respectively). Triglycerides (TGs), cholesterol, and free fatty acid (FFA) concentrations were measured by enzymatic methods (Triglycerides FS, Cholesterol FS, NEFA FS, Diasys). Cytokines were measured with a Milliplex MAP 5-Plex Kit using mouse cytokine/chemokine magnetic bead panel (MCYTOMAG-70K, Millipore), according to the manufacturer's protocol, and using a LuminexR apparatus (Bio-Plex 200, Bio-Rad).

Glycerol and NEFA in plasma or culture medium were measured by colorimetric method using respectively glycerol FS and NEFA FS commercial kits (DiaSys, Grabels, France) according to supplier’s information. Intracellular cAMP levels were determined by a competitive ELISA method using a cAMP Complete ELISA kit (Enzo Life Science, Villeurbanne, France). Briefly, frozen WAT samples were extracted with 0.1 M of HCl in a Mini-Beadbeater (BioSpec Products) and then centrifuged twice at 800 g for 10 min at 4°C. The cAMP assay was performed on the supernatant following the supplier’s instructions.