3h triolein

³H-triolein (#NET431001MC; PerkinElmer, Waltham, MA) was retro-orbitally intravenously injected (2 µCi per mouse in 100 µl of 5% intralipid) into mice after a 16 h fasting. Blood samples (0.15 ml) were then collected at 1, 2, 5, 10 and 15 min after injection. At 15 min following injection, mice were euthanized, blood samples were taken and selective tissues were harvested. Tissues were quickly excised, weighed and frozen in liquid nitrogen and stored at −80 °C until processing. Lipids were then extracted using a chloroform-to-methanol based extraction method^{75 (link)}. The radioactivity content of tissues, including blood samples, was quantified by scintillation counter (Tri-Carb 2910 TR, PerkinElmer, Waltham, MA).

Cells were stained with Bodipy 493/503 (Invitrogen, Carlsbad, CA) for neutral lipids, or DHE (Life Technologies, Grand Island, NY) for superoxide radicals as previously described^{19 (link)}. In parallel, cells and tissues were processed for immunofluorescence with anti-STK25, anti-NFκB, anti-8-oxoG, anti-E06, anti-KDEL, anti-CHOP, or anti-Ki67 antibodies (see Supplementary Table 1 for antibody information). The labeled area was quantified in 6–10 randomly selected microscopic fields (×20) using the ImageJ software (1.47 v; NIH, Bethesda, MD).
To measure β-oxidation, cells were incubated in the presence of (9,10-³H[N])palmitic acid (PerkinElmer, Waltham, MA), and [³H]-labeled water was quantified as the product of free fatty acid oxidation^{14 (link)}. TAG synthesis was measured as previously described^{14 (link)}. Fatty acid uptake was quantified using the Quencher-Based Technology (QBT) Fatty Acid Uptake Assay Kit (R8132; Molecular Devices, San Jose, CA) according to the manufacturer’s recommendations. The TAG hydrolase activity was determined in total cell lysates using [³H]triolein (PerkinElmer) as the substrate in an assay buffer containing 0.25 mmol/l triolein, 0.8 mmol/l phosphatidylcholine, 20 mmol/l Tris, 150 mmol/l NaCl, and 1 mmol/l EDTA, pH 8.0 (Sigma-Aldrich).

Mice were fasted for 4 h and subjected to oral gavage with 10 μl/g body weight of olive oil emulsion containing 2 μCi [³H]-Triolein (Perkin Elmer INC, USA). Liver, epididymal white adipose tissue (WAT), brown adipose tissue (BAT), muscle and heart were harvested after 4 h. In all, 30–50 mg pieces of each tissue were weight and homogenized in PBS. Homogenate [³H]-radioactivity was measured with a scintillation counter (Tri-Carb 2810 TR, PerkinElmer, USA)^{16 (link),55 (link)}.

Bile acid TCDCA and lipase inhibitor Poloxamer 407 were purchased from MilliporeSigma. [³H]Triolein (0.5 mCi/mL) and [¹⁴C]deoxyglucose (0.01 mCi/mL) were purchased from Perkin Elmer. We obtained d-glucose from Merck and human insulin (Humulin R U-100) from Lilly.