Rosiglitazone

Pre-adipocytes were seeded in tissue culture plates coated with 0.1% gelatin (Millipore, Burlington, MA, USA) and grown until confluency at 37°C in a humidified atmosphere with 5% CO2 in complete medium (CM) containing 4.5 g/L glucose DMEM (ThermoFisher Scientific, Waltham, MA, USA), 10% foetal bovine serum, 100 IU/ml penicillin and 100 μg/ml streptomycin. Differentiation of 3T3-L1 cells was initiated two days after reaching confluency (day 0) using CM supplemented with 500 μM 3-isobutyl-1-methylxanthine (IBMX, ThermoFisher Scientific), 1 μM dexamethasone, 1.7 μM human insulin (Actrapid, Novo Nordisk, Bagsværd, Denmark) and 1 μM rosiglitazone (ThermoFisher Scientific). On day 3, cells were switched to CM containing 0.5 μM insulin for 48 hours (day 5). To complete differentiation, cells were cultured in CM with 2% FBS until day 7 and from here onwards maintained on CM containing 1 g/L glucose DMEM (ThermoFisher Scientific) and 2% FBS for two days. Subcutaneous pre-adipocytes were differentiated using a similar protocol until day 5 and from then onwards maintained on 10% FBS medium as previously described [34 (link)]. Mature adipocytes were treated with recombinant murine IL-27 (Biolegend, San Diego, CA, USA) or vehicle control (PBS supplemented with 1% BSA) as indicated.

The Green PolarScreen PPARγ Competitor Assay (ThermoFisher Scientific, Waltham, MA, USA), which is based on the reactivity of the binding domain of recombinant human PPAR-γ (peroxisome proliferator-activated receptor gamma) with a specific fluorescent PPAR-γ ligand, was used according to the manufacturer’s protocol. The PPAR-γ/PPAR-γ ligand complex emits fluorescence, which is reduced in the presence of an unlabeled competitor. The cinnamon fraction (final concentrations of 15, 30, and 60 μg/mL) was tested as a potential competitor. Rosiglitazone (40 μM; Thermo Fisher Scientific) was used as a positive unlabeled competitor control. Fluorescence was measured using a Synergy 2 microplate reader with the excitation and emission wavelengths set at 485 and 528 nm, respectively.

Mouse 3T3-L1 pre-adipocytes were cultured in the growth medium (GM), composed of Dulbecco’s modified Eagle medium (DMEM; Invitrogen, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (FBS; Invitrogen). To induce in vitro browning, 3T3-L1 cells were shifted to the induction of DMEM medium, supplemented with 20% FBS, 0.5 mM IBMX (Invitrogen), 12.7 μM dexamethasone (Invitrogen), and 10 µg/mL insulin (Invitrogen) for 48 h. The differentiating DMEM medium (DM) supplemented with 10% FBS, 10 μg/mL insulin, and 2 µM Rosiglitazone was replaced with the induction medium and replenished every 48 h for 4 days. BBR, purchased from Sigma–Aldrich (St. Louis, MO, USA), was dissolved in DMSO. 3T3-L1 cells maintained in the growth medium were treated with 5 μM BBR for 48 h.

Cells were cultured in adipogenic induction medium (DMEM-Ham’s F12, 3% FBS, 1% antibiotic solution, biotin (33 mmol/L), pantothenate (17 mmol/L), insulin (1 mmol/L), dexamethasone (1 mmol/L), isobutylmethylxanthine (IBMX, 0.5 mmol/L), rosiglitazone (5 mmol/L) (TZD, AK Scientific, Union City, CA), 5% rabbit serum (Invitrogen Corporation, Carlsbad, CA)) for 3 d followed by adipogenic maintenance medium (adipogenic medium minus IBMX and rosiglitazone) for 2 d. The presence of intracellular lipids was confirmed by staining with oil red O for 20 min after cells were fixed overnight in 4% paraformaldehyde at room temperature and then washed with PBS. An inverted phase contrast microscope (Olympus® CKX41SF, Japan) instrumented with a digital camera (Olympus DP21, Japan) was used to obtain digital images.

At full confluence, PDGFRα⁺ cells were exposed to adipogenic medium consisting of low-glucose DMEM supplemented with 10% FBS (Gibco), 1 mM dexamethasone (D1756, Sigma-Aldrich, St. Louis, MO, USA), 0.5 mM isobutyl-methylxanthine (I5879, Sigma-Aldrich, St. Louis, MO, USA), 3 μg/mL of insulin (I5500, Sigma-Aldrich, St. Louis, MO, USA), 1 mM rosiglitazone (R2408, Sigma-Aldrich, St. Louis, MO, USA) and 100 U/mL of penicillin-streptomycin (Gibco). After two days, the cells were switched to maintenance medium consisting of low-glucose DMEM (Gibco) supplemented with 3 μg/mL of insulin, 1 mM rosiglitazone, and 100 U/mL of penicillin-streptomycin (Gibco).