Cell culture and treatment

3T3-L1 cells were chosen in the present study for their extensive use in evaluating the effects of compounds or nutrients on adipogenesis and in the potential application of various compounds and nutrients in the treatment of obesity. Moreover, at the best of our knowledge, the browning effect of BAIBA on 3T3-L1 cell model was never investigated. The murine 3T3-L1 preadipocytes (ZenBio Inc., Durham, NC, USA) were grown in high glucose DMEM supplemented with 10% FBS, 1% amphotericin B solution and 1% penicillin/streptomycin solution, at 37 °C with 5% CO₂ and 95% relative humidity. Differentiation was induced 48 h after cells reached full confluence with DMEM/F-12 medium supplemented with 10% FBS, 1% amphotericin B solution, 1% penicillin/streptomycin solution, 0.5 μg/mL human insulin, 5 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 1 nM triiodothyronine (T₃) and 1 μM rosiglitazone. After 3 days, the differentiation medium was switched to a maintenance medium to which 0.5 μg/mL human insulin and 1 nM T₃ were added. Maintenance medium was refreshed every 2 days. After the switch from differentiation to maintenance medium, cells were treated with 1, 3 and 5 μM BAIBA until analysis. Cells were analyzed during differentiation at day 4 (4d) and at day 8 (8d), at day 10 (10d); these time points correspond to 2, 6 and 8 days of BAIBA treatment, respectively. A negative vehicle control (CTRL) was established treating cells with sterile milliQ water.