Fatty acid oxidation

H4IIE hepatocytes were allowed to adhere to 24-well culture plates XF24 cell culture microplate (Seahorse Bioscience, Cat# 100777–004) after seeding for 2 days, according to standard protocols (Seahorse Bioscience). Cells were pre-incubated with 5 μg/ml CTRP3 or vehicle for 1 H then transferred into XF assay medium (Seahorse Bioscience, Cat# 100965–000) supplemented with 0.5mM sodium pyruvate and 5mM glucose before being placed into the XF24 Extracellular Flux Analyzer (Seahorse Bioscience XF24). The dosage of recombinant CTRP3 was selected based on our previous experimental observations [9], and is well within the common dosages reported within the literature of 2–30 μg/ml [9, 29, 43, 44]. Sensor cartridge of XF24 extracellular flux assay kit (Seahorse Bioscience, Cat# 100850–001) was hydrated by loading 1ml XF calibrant (Seahorse Bioscience, Cat# 100840–000) into each well in utility plate and incubating at 37°C overnight in a non-CO₂ incubator. 200uM palmitate conjugated to bovine serum albumin (BSA) or fatty acid free BSA (vehicle control) was added in XF24 cell culture microplate at time-points indicated. A 1 mM working stock of palmitate (Sigma Cat# P5585) conjugated to 0.17 mM fatty acid free BSA (Sigma Cat# A8806) was prepared according to established protocols (Seahorse Bioscience). Briefly, 50 ml of a BSA solution (0.34 mM BSA, 150 mM NaCl, pH 7.4) was heated to 37 C and 40 ml of a palmitate solution (2.98 mM palmitate, 150 mM NaCL), heated to 70 C, and was added in 5 mL increments. The combined solution was incubated at 37 C for 1 H under constant agitation, afterwards the pH was adjusted to 7.4 and final volume was adjusted to 100 mL with 150 mM NaCl). Aliquots were stored until use at -20 C in glass vials.