1 14c pyruvic acid

Oleic acid oxidation and incorporation into triglycerides were assessed as described in Ref. [41] (link). To determine pyruvate oxidation, insulin was removed from culture medium the day before assay. Cells were incubated for 3 h in 1 ml Krebs–Ringer Buffer (125 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1.25 mM KH₂PO₄, 1.25 mM MgSO₄, 25 mM NaHCO₃) supplemented with 2% BSA, 10 mM HEPES, 2 mM glucose, 1 mM pyruvate and 0.5 μCi [1-¹⁴C]-pyruvic acid (PerkinElmer) with or without 100 nM insulin. Medium was then acidified with 1 M sulfuric acid in closed vials containing a central well filled with benzethonium hydroxide. After 3 h incubation, trapped ¹⁴CO₂ was measured by liquid scintillation counting. Specific activity was measured and used to determine the quantity of oxidized pyruvate equivalent. Cells were washed with PBS and scraped in STED buffer (0.25 M sucrose, 10 mM Tris–HCl,1 mM EDTA, 1 mM dithiothreitol, pH 7.4). Results were normalized to protein content determined with Bio-Rad protein assay using BSA as standard.