Stromal vascular cells were obtained from iBAT and iWAT excised from 3-week-old C57BL6 mice (males and females), primary cultures were generated and the cells were induced to differentiate into brown and beige adipocytes, respectively, following the previously reported procedures25 (link)26 (link). Brown adipocyte differentiation was achieved by exposing confluent precursor cells from iBAT in DMEM/F12 medium containing 10% foetal bovine serum (FBS) and supplemented with 20 nM insulin, 2 nM T3 and 0.1 mM ascorbic acid (ITA).
For beige cell differentiation, confluent precursor cells from iWAT and eWAT were maintained in DMEM/F12 containing 10% newborn calf serum (NCS). For differentiarion, 850 nM insulin, 3 μM T3, 35 nM dexamethasone and 10 μM rosiglitazone were added. When indicated, pre-adipocytes were cultured in the presence of delipidated serum (Charcoal Stripped Serum-GIBCO) instead of FBS/NCS
Cells were treated either across the differentiation process or were treated acutely (24 h), when already differentiated. Treatments included TUG-891 (200 μM), grifolic acid (100 μM), GW9508 (100 μM), ALA (100 μM), EPA (100 μM), NE (0.5 μM), dibutyryl-cAMP (1 mM), SB202190 (10 μM), H89 (20 μM), GW7647 (1 μM), GW9662 (30 μM), AH-7614 (100 μM), U-0128 (10 μM), compound C (10 μM), wortmannin (2 μM) and CL316,243 (1 μM). All reagents were obtained from Sigma with the exception of TUG-891, AH-7614, GW9662 (from Tocris), GW9508 (from Cayman Chemical), compound C (Calbiochem) and U-0128 (Enzo). When indicated, cells were subjected to dynamic measurements (see below) and/or further collected for RNA extraction.
For beige cell differentiation, confluent precursor cells from iWAT and eWAT were maintained in DMEM/F12 containing 10% newborn calf serum (NCS). For differentiarion, 850 nM insulin, 3 μM T3, 35 nM dexamethasone and 10 μM rosiglitazone were added. When indicated, pre-adipocytes were cultured in the presence of delipidated serum (Charcoal Stripped Serum-GIBCO) instead of FBS/NCS
Cells were treated either across the differentiation process or were treated acutely (24 h), when already differentiated. Treatments included TUG-891 (200 μM), grifolic acid (100 μM), GW9508 (100 μM), ALA (100 μM), EPA (100 μM), NE (0.5 μM), dibutyryl-cAMP (1 mM), SB202190 (10 μM), H89 (20 μM), GW7647 (1 μM), GW9662 (30 μM), AH-7614 (100 μM), U-0128 (10 μM), compound C (10 μM), wortmannin (2 μM) and CL316,243 (1 μM). All reagents were obtained from Sigma with the exception of TUG-891, AH-7614, GW9662 (from Tocris), GW9508 (from Cayman Chemical), compound C (Calbiochem) and U-0128 (Enzo). When indicated, cells were subjected to dynamic measurements (see below) and/or further collected for RNA extraction.
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