Adipocytes, Brown

TERT-hBA and TERT-hWA cells were seeded at 1 × 10⁶ per well in 6-well plates. At two-day post confluence (designated day 0), adipogenesis was induced with a differentiation cocktail containing Advanced DMEM/F12 containing 2% FBS, 2 mM L-glutamine, 62.5 μg/mL penicillin, 100 μg/mL streptomycin (basal medium), and supplemented with insulin (5 μg/mL), dexamethasone (1 μM), 3-isobutyl-1-methylxanthine (IBMX) (0.5 mM), rosiglitazone (1 μM), human cortisol (1 μM), and T3 (1 nM). On day 3, the medium was refreshed with the same medium used at day 0. Between days 6 and 12, TERT-hWA were cultured in basal medium to reach full white differentiation (day 12). For “browning” of white adipocytes, TERT-hWA were further cultured for 3 days, until day 15, in the presence of 1 μM rosiglitazone. Conversely, between days 6 and 12 TERT-hBA were differentiated to brown adipocytes by addition of 1 nM T3 to the basal culture medium [17 (link)]. Table S4 in the supplementary data summarizes the differentiation protocol. At day 6, approximately 60% of cells already showed an adipocyte morphology, with accumulation of lipid droplets, while at day 10 this percentage increased to about 80–90%. During white or brown differentiation, TERT-hBA and TERT-hWA cells were treated or not (controls) with 100 nM ABA from day 0 to day 12; during the “browning” process of white adipocytes (days 12–15), differentiated TERT-hWA cells were treated with or without 100 nM ABA, 1 μM rosiglitazone or both.

Murine HIB1B brown preadipocytes were differentiated to mature brown adipocytes as previously described [57 (link)]. Briefly, HIB1B cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum and 1% penicillin/streptomycin at 5% CO₂ and 37 °C. For differentiation, cells were cultured to confluence (day 0) and then exposed to differentiation medium 1 (0.5 mM IBMX, 0.25 µM dexamethasone, 20 nM insulin, 1 nM T3). After 48 h, cells were maintained in differentiation medium 2 containing 20 nM of insulin and 1 nM of T3. At five time points during brown adipocyte differentiation (days 0, 1, 3, 5, 7), cells were harvested for RNA isolation and on day 7 for Oil-Red O staining.
Murine 3T3-L1 cells were differentiated to mature white adipocytes as previously described [58 (link)]. Briefly, 3T3-L1 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum and 1% penicillin/streptomycin at 5% CO₂ and 37 °C. For differentiation, cells were seeded into 6-well plates (100,000 cells/well), cultured to confluence (day 0) and treated with differentiation medium containing 0.5 mM of IBMX, 1 µg/mL of insulin and 0.25 µM of dexamethasone. The culture medium was then changed on day 5, 7 and 10 to maintenance medium supplemented with 1 µg/mL of insulin. For the induction of white adipocyte browning, media were either supplemented with 2 µM of rosiglitazone or 8.3 nM of human BMP7 (R&D Systems, Minneapolis, MN, USA) during the whole period of adipocyte differentiation. Cells were harvested before adipogenic induction (day 0) and on days 4, 8 and 12 after adipogenic induction for RNA and protein isolation, and on day 12 for Oil-Red-O staining.