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Figure 1. Orlistat inhibits the thioesterase domain of fatty acid synthase and inhibits the metabolic function of FAS in human umbilical vein endothelial cells. A) The effect of orlistat on the FAS thioesterase was probed in HUVEC lysates that were treated with orlistat (0–10 µM) or vehicle control for 30 min. The treated lysates were exposed to the activity-based probe, FP-TAMRA (5 µM), resolved using SDS-PAGE and visualized by scanning with a Hitachi fluorescent gel scanner. B) Lysates treated with orlistat or control and exposed to FP-TAMRA (5 µM) were immune-precipitated with antibodies against FAS and resolved using SDS-PAGE. Bands were visualized by scanning with a Hitachi fluorescent gel scanner and the gel was subsequently transferred onto nitrocellulose paper and immunoblotted with antibodies against FAS. C) The effect of orlistat on the incorporation of [14C] malonyl CoA was measured in lysates of HUVECs. The lysates, containing FAS, were treated with orlistat or vehicle for 1 h. The lysates were subsequently incubated with [14C] malonyl CoA for 25 min at 37°C to allow the malonyl CoA to incorporate into palmitate. Fatty acids were extracted from each sample and quantified by scintillation counting. Triplicate wells were assigned for each condition and statistically significant differences were observed between control treated lysate and each of orlistat-treated lysate (P<0.2 for 0.25 µM and P<0.05 for 0.5 µM). D) The effect of orlistat on the incorporation of acetate into fatty acids was probed in live HUVECs. The cells (1x105 cells/well in 24-well plates) were cultured in EGM-MV for 24 h and subsequently serum-starved for 16 h in EBM containing VEGF (40 ng/ml; Chemicon International, Temecula, CA). Serum-starved cells were treated with 15 µM orlistat for 4 h. [14C] acetate was added to each well and incubated for 3 h at 37°C. After incubation with [14C] acetate, fatty acids were extracted and quantified by scintillation counting. Six wells were designated as control + VEGF; three wells were designated as orlistat + VEGF. Statistically significant differences were observed between these two conditions (P<0.05). Three wells were grown without VEGF as growth control. E) The effect of orlistat on the proliferation of HUVECs was determined by measuring the incorporation of BrdU following treatment of cells with the drug. HUVECs were seeded in 96-well plates at a density of 2000 cells/well and cultured in endothelial growth media for 24 h at 37°C. Cells were subsequently cultured in media containing EBM and 2% FCS for 24 h. Culture media was replaced with EBM containing 2% FCS and VEGF (20 ng/ml) to stimulate proliferation. At this time cells were treated with orlistat (0–40 µM) and BrdU (100 µM) was added to track DNA synthesis. After a 24-h incubation the incorporation of BrdU uptake was measured using colorimetric Cell Proliferation ELISA (Roche) as described in Materials and Methods. Triplicate wells were used for each condition. SE in three repetitions of this experiment was typically 10%. F) The effect of orlistat on endothelial cell death was also measured across a concentration range of the drug. HUVECs were grown to 60% confluency (proliferating •) or 100% confluency (contact-inhibited 
) and then exposed to orlistat for 48 h. Cell death was measured with the Roche Cell Death ELISA kit. Each point is the average of triplicate data points. SE was less than 12% for each point.
