Glycerol fs

In vivo experiments. Overnight-fasted mice were injected i.p. with JM-00266 (mg·kg⁻¹) or vehicle. Four hours later, mice were injected i.p. with the specific ß3-adrenergic receptor agonist, BRL37344 (5 mg·kg⁻¹). Blood samples (~25 µL) were collected from tails in tubes containing EDTA (Sarstedt, Nümbrecht, Germany) at times 0, 15, 30, 45 and 60 min after BRL37344 injection to measure plasma glycerol appearance using a colorimetric assay (glycerol FS, DiaSys, Condom, France).
In vitro experiments. Overnight-fasted mice were injected i.p. with JM-00266 (mg·kg⁻¹) or vehicle. Four hours later, mice were anesthetized with pentobarbital (50 mg·kg⁻¹) and epididymal adipose tissue was collected, rinsed in a warm medium (DMEM-HAM/F12) supplemented with 1% BSA, and dissected into ~10 mg pieces. Then, 5–6 explants were incubated in a 5% CO₂ atmosphere at 37 °C under slight agitation in 1 mL of the same medium. Free glycerol in the culture medium was determined after 1 h of incubation using a colorimetric assay (glycerol FS, DiaSys).

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.