Acetyl coa

The chemical compound library was from Open Innovation Center for Drug Discovery, University of Tokyo (Tokyo, Japan) (23 ). The library includes 174,131 compounds supplied as 10 or 2 mM solutions in DMSO. After the selection of N-phenylmaleimide derivatives, each compound was obtained in powdered form from commercial vendors (see supplementary Methods). Methylcarbamyl-PAF (mcPAF, nonhydrolyzed analog of PAF), 16:0 PAF, 16:0 lyso-PAF, deuterium-labeled (d₄)-16:0 PAF, d₄-16:0 lyso-PAF, and d₃₁-16:0 lyso-PC were from Cayman Chemical Company (Ann Arbor, MI). DPPC standards were purchased from NOF Corporation (Tokyo, Japan). Palmitoyl-CoA and arachidonoyl-CoA were from Avanti Polar Lipids (Alabaster, AL). Acetyl-CoA, DMSO, chloroform, and LC-MS grade solvents (methanol and acetonitrile) were from Wako (Osaka, Japan). Lipopolysaccharide (LPS) from Salmonella minnesota was purchased from Sigma-Aldrich (St. Louis, MO), and A23187 (calcium ionophore) was from Biomol (Plymouth Meeting, PA). Two types of protease inhibitor cocktails (complete and EDTA-free complete) were purchased from Roche Applied Science (Mannheim, Germany).

CS activity was determined using previously published methods (Srere & Bhaduri, 1964 (link)). In summary, supernatants were solubilized in a reaction buffer comprising 0.1 mM DTNB (D8130‐500MG, Sigma‐Aldrich, St. Louis, MO) and 0.3 mM acetyl‐CoA (014–10,813, Wako Pure Chemical Industries). The reaction was initiated with 0.5 mM oxaloacetic acid (014–10,813, Wako Pure Chemical Industries), and absorbance was monitored at 412 nm for 5 min.

The activity of citrate synthase (CS) was measured as previously reported, with some modifications [55 (link)]. Briefly, WAT samples were homogenized in homogenization buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% phosphatase inhibitor cocktail, 5 mM EDTA, 1% protease inhibitor cocktail, 1% Triton X-100, and 0.05% sodium deoxycholate. Protein concentration was determined using a BCA protein assay kit (Thermo Scientific, Rockford, IL, USA), according to the manufacturer’s protocol. For CS activity measurements, a reaction mixture containing 0.1 mM 5,5-dithiobis-(2-nitrobenzoic) acid (Wako), 0.5 mM acetyl-CoA (Wako), 0.1% Triton X-100, and 100 mM Tris-HCl, pH 8.0, was used with the tissue homogenates, which contained 5–8 mg protein. After incubation at 25 °C for 5 min, the absorbance at 412 nm (SpectraMax Plus384, Molecular Devices, Sunnyvale, CA, USA) was measured over 3 min to determine the non-specific activity. Reactions were then initiated by addition of 0.5 mM oxaloacetate (Wako) in a final volume of 200 mL, and the change in absorbance was recorded for at least 3 min.