Sodium palmitate p9767

MPC1 (14462), MPC2 (46141), β-Actin (4970), Akt (9272) were purchased from Cell Signaling Technologies, UCP1 (AB10983), Cytochrome C [7H8.2C12] (AB13575), HMGB1 (AB18256) were purchased from Abcam. 4-hydroxy-tamoxifen (4-OHT) and UK5099 were purchased from Tocris. CL-316,243 (C5796) was purchased from Sigma. U-¹³C D-Glucose (CLM-1396–5) and U-¹³C Sodium palmitate (CLM-6059–1) were purchased from Cambridge Isotopes. Sodium palmitate (P9767) was purchased from Sigma Aldrich. DL-[1-¹⁴C] 3-hydroxybutyric acid sodium salt (ARC1455) was purchased from American Radiolabeled Chemicals. DL-β-Hydroxybutyric acid sodium salt (H6501) was purchased from Sigma.

Bone marrow cells were isolated from femurs of 3–6 months old male TG2 null mice and their wild type counterparts. Bone marrow macrophages (BMDMs) were differentiated in DMEM supplemented with 10% FBS (12106C), 2 mM L-glutamine (G7513), 1 mM Na-pyruvate (S8636), 50 μM 2-mercaptoethanol (M3148) and 100 U/ml penicillin/100 μg/ml streptomycin (P4333) all from Sigma-Aldrich and 10% L929 fibroblast conditioned media for 7 days. For metabolic activation, differentiated macrophages were treated with a combination of 30 mM D-glucose (G8270), 10 nM insulin (12643) and 0.4 mM sodium-palmitate (P9767) all from Sigma-Aldrich for 24 h (16). Sodium palmitate was prepared by diluting a 200 mM stock solution in 70% ethanol into 10% fatty acid-free, low-endotoxin BSA (Sigma Aldrich, A8806 adjusted to pH 7.4) to obtain a 5 mM palmitate-BSA stock solution that was filtered using a 0.22-μm low-protein binding filter (Millipore). BSA/ethanol was used in control treatments during the protocol. In some experiments during the 24 h metabolic activation BMDMs were treated with PP2 (Sigma Aldrich, 529573), a reversible ATP-competitive inhibitor of the Src family of protein tyrosine kinases, in 2 µM final concentration or 0.5 mg/ml RGD peptide (Cayman Chemical, 529573).

Oleic acid (O1008) and sodium palmitate (P9767) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). A 100 mM oleic acid (OA) stock solution was prepared in 0.1 M NaOH by heating at 70°C in a shaking water bath (Cousin et al., 2001 (link)). In an adjacent water bath at 55°C, the 100 mM OA stock solution was dissolved at 5 mM in culture medium containing 1% bovine serum albumin. Sodium palmitate (PA) was dissolved at 100 mM in distilled water, shaken at 70°C, dissolved at 5 mM in culture medium containing 1% bovine serum albumin, and then shaken at 37°C. Both solutions of OA and PA were stored at 4°C. The final concentration of OA- and PA-induced cells was 50 μM, and the medium contained 0.01% BSA.