3h palmitic acid

Cultured cells were washed with HBSS and incubated with 200 µL of [³H]-palmitic acid (1 mCi/mL, PerkinElmer) bound to fatty-acid free albumin (100 µM; the ratio of palmitate:albumin is 2:1) and 1 mM l-cartinine. The complex was incubated for 2 h at 37 °C. The supernatant was collected after incubation and added to a tube containing 200 µL of cold 10% trichloroacetic acid. The tubes were centrifuged 10 min at 3000g at 4 °C, and aliquots of supernatants (350 µL) were removed, neutralized with 55 µL of 6 N NaOH and applied to an ion exchange column loaded with Dowex 1X2 chloride form resin (Sigma-Aldrich). The radioactive product was eluted with water. Flow-through was collected and radiation was quantified using liquid scintillation counting.

Fetal bovine serum, penicillin/streptomycin, and DMEM were purchased from Invitrogen. PAO, rapamycin, and SP600125 were obtained from Calbiochem, and [3H]-palmitic acid was from Perkin Elmer Life Sciences. 1-palmitoyl-2-arachidonoyl-sn-glycerol-3-phosphate (PA) dissolved in chloroform was purchased from Avanti Polar Lipids (Alabaster, AL, USA). The following polyclonal antibodies from Cell Signaling (Beverly, MA, USA) were used: PLD1, PLCγ, p-PLCγ, Src kinase, p-Src kinase, mTOR, p-mTOR, p-p70S6K antibody, and p70S6K. GAPDH and PLCγ were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Transfected cells were incubated for 4 h in medium containing 0.5mCi/ml [³H]palmitic acid (Perkin Elmer) and 0.1% BSA (fatty acid free). The GFP-tagged proteins were then immunoprecipitated with magnetic microbeads coupled to GFP antibody (Miltenyi Biotech). Immunoprecipitated proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. [³H]palmitate present on the recovered GFP-tagged proteins was detected using a Kodak Biomax Transcreen LE intensifier screen.

Sciatic nerve and whole brain were removed and split in half for 2× cortex and 2× cerebellum pieces. Tissues were then weighed and placed in KH buffer containing 25 mM NaHCO₃, 118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO₄, 1.2 mM NaH₂PO₄, 1.2 mM CaCl₂ and 2.5 mM glucose. Tissues were kept in buffer on ice until all dissections were completed. Each tissue then transferred to KH buffer plus 2% Fatty acid free BSA and 2.5 mM glucose, with 2 μCi 3H palmitic acid (Perkin Elmer, Waltham, MA, USA) and incubated at 37°C for 1 hr. The buffer was collected and hydrolyzed 3H palmitic acid (as 3H water) was extracted. 100 μL of buffer was added to 100 μL of 10% trichloroacetic acid (TCA), vortexed, incubated at RT for 15 min, spun at 16,000 rpm for 10 min, and the supernatant collected into a new tube. TCA (5%; 100 μL) and 40 μL BSA (10%) was added to the supernatant, vortexed, incubated at RT for 15 min, spun at 16,000 rpm for 10 min, and the supernatant transferred to a new tube. Chloroform:methanol (2:1, 750 μL) was added to the supernatant, along with KCl: HCl (2 M each, 300 μL), vortexed and spun at 16,000 rpm for 10min. The upper layer (~600 μL) was collected into 5 ml EcoLume, mixed, and counted in a liquid scintillation counter. After subtracting background cpm, the sample cpm was divided by the tissue weight to determine fatty acid oxidation capacity.

At initiation of treatment, [³H]-palmitic acid (1 μCi/ml medium) purchased from Perkin Elmer (32.0 Ci/mmol) was added to treated and untreated samples. At the indicated time points, lipids were extracted according to Bligh and Dyer method [49 (link)], N₂ dried, and resuspended in 60 μl chloroform:methanol (2:1); 40 μl were spotted on 20 cm silica gel TLC plates. Plates were developed with ethylacetate:isooctane:acetic acid (90:50:20, v/v), air dried, and sprayed lightly with En3hance® (Perkin Elmer) to enhance tritium readings. [³H]-Cer spots were visualized by iodine vapor mark. Radioactivity was visualized by autoradiography after 96 h at −80°C and the [³H]-Cer spots were scraped into scintillation vials containing 4 ml of scintillation fluid and counted on a Packard scintillation counter. [³H]-Cer counts were normalized to lipid phosphate levels.