9 10 3h n oleate

FFA and glucose uptake were assessed in mice that were injected with PBS or P407 and then fasted for 16 h. [9,10-³H(N)]oleate (PerkinElmer Life Sciences) was complexed to 6% FA-free BSA (Sigma). Mice were injected intravenously with 1 μCi [9,10-³H(N)]oleate-BSA and blood was collected at 0.5, 1, 3, and 5 min after injection. Five minutes after injection, the body cavity was perfused with 10 ml of PBS by cardiac puncture and tissues were excised. Tissue was homogenized in PBS and radioactive counts were measured. Basal glucose uptake was measured in hearts following an intravenous administration of 2.5 μCi of 2-deoxy-D-[1-¹⁴C]glucose (PerkinElmer Life Sciences). Blood was collected 2, 30, and 60 min following injection. At 60 min, hearts were perfused with PBS, tissues were excised, and radioactive counts were measured. For all turnover studies, radioactivity per gram of tissue was normalized to the respective 30 s or 2 min plasma counts (injected dose).

Fully differentiated adipocytes were incubated with 0.1 mM palmitate/oleate/linoleate (1:2:1) lean or 1 mM palmitate/oleate/linoleate (1:2:1) obese DMEM media supplemented with [9,10-³H(N)]-oleate (0.5 μCi/ml, Perkin Elmer Inc., USA) in 2% BSA for about 24 h. Specific activity was determined by measuring cellular TAG content (as above), and ³H in the TAG pool was assessed by a Folch extraction of cellular lipids followed by thin layer chromatography [27 (link)]. After incubation, adipocytes were co-cultured with either MCF-7 or MDA-MB-231 cells that were pre-seeded 1 × 10⁵ cells/ well for 24 h. MCF-7 and MDA-MB-231 cells were scraped in PBS and ³H activity determined by liquid scintillation counting.