反应详情

Lipolysis of 3T3-L1 cells. 3T3-L1 fibroblasts (CL-173) were obtained from ATCC (Teddington, UK) and cultivated in DMEM containing 4.5 g/liter glucose and L-glutamine (Invitrogen) supplemented with 10% FCS and antibiotics under standard conditions. Cells were seeded in 12 well plates and two days after confluence, medium was changed to DMEM supplemented with 10% FCS containing 10 μg/ml insulin (Sigma-Aldrich), 0.25 μM (0.4 μg/ml) dexamethasone (Sigma-Aldrich), and 500 μM isobutylmethylxanthine (Sigma-Aldrich). After 3 and 5 days, medium was changed to DMEM supplemented with 10% FCS containing 10 μg/ml and 0.5 μg/ml insulin, respectively. On day 7 of differentiation the cells were incubated ON in the absence of insulin. Cells were used at day 8 of differentiation. Therefore cells were preincubated with 0, 0.1, 1, 10, or 50 μM of Atglistatin in the presence or absence of 10 μM Hi 76-0079 (NNC 0076-0000-0079, provided by Novo Nordisk, Denmark) for 2 h. Then, the medium was replaced by DMEM containing 2% BSA (fatty acid free, Sigma, St. Louis, Mo.), 10 μM forskolin, and 0, 0.1, 1, 10, or 50 μM of Atglistatin in the presence or absence of 10 μM Hi 76-0079 for 1h. The release of FFA and glycerol in the media was determined using commercial kits (NEFA C, WAKO, free glycerol reagent, Sigma). Protein concentration was determined using BCA reagent (Pierce) after extracting total lipids using hexane:isopropanol (3:2), and lysing the cells using SDS:NaOH (0.3%:0.1 N).

1. customwere seeded in 12 well plates and two days
2. customAfter 3 and 5 days, medium
3. additioncontaining 10 μg/ml and 0.5 μg/ml insulin
4. waitOn day 7 of differentiation the cells were incubated ON in the absence of insulin
5. waitCells were used at day