Dexamethasone (dex)

Reagents and standards, including acarbose, acetonitrile, α-amylase, α-glucosidase, camphor, cinnamaldehyde, cinnamic acid, dexamethasone, dimethyl sulfoxide (DMSO), eugenol, formic acid, glucose, glutaMAX, insulin, 1-isobutyl-3- methylxanthine, methanol, menthol, p-nitrophenyl-α-D-glucopyranoside (PNPG), hydroxyethyl piperazine ethane sulfonic acid (HEPES), and resazurin sodium were bought from Sigma-Aldrich, USA. Glycyrrhizic acid monoammonium salt was obtained from Carlo Erba (Italy). Fetal bovine serum (FBS), Dulbecco's modified Eagle's medium (DMEM), antibiotic/antimycotic solution, Roswell Park Memorial Institute 1640 medium (RPMI 1640), 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA), and Qubit dsDNA broad range (BR) assay kits were obtained from Thermo Fisher Scientific, USA. A C-peptide 2 (Rat/Mouse) ELISA kit and phosphate-buffered saline solution (PBS) were bought from Merck-Millipore. A Glucose Uptake Assay kit (cell-Based) was acquired from Cayman Chemical (USA). The cell lines 3T3 J2 fibroblasts, 3T3-L1 fibroblasts, and RIN-m5F cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

C3H10T1/2 cells (ATCC, CCL-226) were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, C11995500BT) with 10% fetal calf serum (Selborne, CS-05) and 1% Penicillin/Streptomycin solution (Invitrogen) in 10% CO₂ at 37°C. The cells were maintained until fully confluent and then induced to differentiate in DMEM containing 10% fetal bovine serum (FBS, Gibco, 16000-044), 0.5 mM 3-isobutyl-1-methylxanthine (IBMX, Sigma-Aldrich, I7018), 1 μM dexamethasone (Sigma-Aldrich, D4902), 10 μg/mL insulin (Roche, 11376497001) and 1 μM rosiglitazone (Sigma-Aldrich, R2408) for two days, and then the cells were cultured in DMEM (10% FBS, 10 μg/mL insulin and 1 μM rosiglitazone) for an additional 2 days. After these procedures, the cells were maintained in DMEM containing 10% FBS. To obtain mouse primary adipocytes, SVFs isolated from iWAT or eWAT of 4-week-old C57BL/6 J male mice were maintained in DMEM/F12 (Gibco, 11330033) containing 10% FBS until post-confluence. Then cells were induced to differentiate with the same procedure as C3H10T1/2. Human primary adipocytes isolated from human subcutaneous adipose tissue (sex: male; 24 < BMI < 28) (ChiCTR2300074800) were cultured in DMEM/F12 with 20% FBS until reaching post-confluence. Then cells were induced to differentiate into mature adipocytes with 1 μM rosiglitazone, 0.5 mM IBMX, 1 μM dexamethasone and 1 μg/mL insulin for 2 days before being incubated in DMEM/F12 containing 10% FBS, 1 μM rosiglitazone and 1 μg/mL insulin for another 2 days. This induction procedure was repeated several times until lipid droplets appeared. HEK293T was purchased from ATCC (CRL-1573). Huh7 (SCSP-526) and Hela (SCSP-504) were obtained from the National Collection of Authenticated Cell Cultures. These cells were cultured in a 37°C incubator with 5% CO₂ and maintained in DMEM supplemented with 10% FBS (Gibco) and 1% Penicillin/Streptomycin solution (Invitrogen). Mycoplasma contamination was tested in all cells at least once a month and consistently proven to be negative.