14c oleate

TG synthesis was measured as described before^{18 (link)}. Briefly, synthesis of triglycerides derived from [¹⁴C]-oleate (PerkinElmer) complexed with BSA was measured by incubation of untreated or OL-treated cells with [¹⁴C]-oleate-BSA (0.2 μCi, 4 h), followed by extraction of cellular lipids and analysis by thin-layer chromatography. Incorporation of radioactivity into TG was determined using a Storm Gel and Blot Imaging System (GE Healthcare) followed by densitometry using ImageJ. The rate of TG synthesis was calculated as the amount of incorporated [¹⁴C]-oleate in relative units per mg protein.

Fatty acid uptake assay kit (QBT, Molecular Devices) that utilizes fluorescent bodipy-C₁₂, a LCFA analog, was used to determine fatty acid uptake^{50 (link)}. Differentiated 3T3-L1 adipocytes were plated at 5 × 10⁴ cells per well in a 96-well plate. Adipocytes were first incubated in serum-free Hanks balanced salt (HBS) solution for 4 h, and then with 100 nM insulin and bodipy-C₁₂ for an additional 4 h. After 30 min, relative fluorescence was read at 485 nm excitation and 515 nm emission wavelength in bottom-read mode (SpectraMax GeminiEM, Molecular Devices).
LCFA uptake was also determined in differentiated 3T3-L1 adipocytes as cellular accumulation of [¹⁴C]oleate (Perkin-Elmer). Adipocytes (10,000 cells) were seeded in a 24-well plate in DMEM with 10% calf serum overnight. Cells were serum-deprived for 4 h, treated with 100 nM insulin for 3.5 h, and then with 50 μM of [¹⁴C]oleate in HBS containing 0.1% fatty acid-free BSA for 30 min^{51 (link),52 (link)}. Cells were washed extensively in cold HBS with 0.1% fatty acid-free BSA to remove unincorporated [¹⁴C]oleate, lysed in RIPA buffer (Thermo Fisher), and centrifuged at 2000 rpm for 5 min. Supernatant radioactivity was determined by scintillation counting and normalized to protein. LCFA uptake by mouse WAT, hepatocytes, cardiac cells, BMDM, and soleus muscle strips were measured using essentially the same method^{13 (link)}.