1 14c oleate

Fatty acid-free BSA, myristic acid, oleic acid, palmitic acid, and 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB) were purchased from Sigma-Aldrich (St. Louis, MO). Antibiotics, high-density nickel resin, and IPTG were purchased from GoldBio (St. Louis, MO). [1-¹⁴C]acetate (55 mCi/mmol) and [1-¹⁴C]oleate (56.3 mCi/mmol) were purchased from PerkinElmer (Waltham, MA). Bacteria media supplies were purchased from BD Medical Technologies (Franklin Lakes, NJ). All chemicals and solvents were reagent grade or better.

All chemicals and reagents were reagent grade or better. [¹³C₆]Isoleucine, [d₃]leucine, [¹³C₅,¹⁵N]valine were purchased from Cambridge Isotope Laboratories, Inc. DSC-NH₂ SPE columns (Supelco, Sigma-Aldrich), Bee venom PLA₂ (P9297-Sigma), Orlistat (O4169-Sigma), [1-¹⁴C]oleate (specific activity 59 mCi/mmol) (Perkin Elmer).

The fatty acid oxidation assay followed the protocol described in [31 ], adapted from [35 (link)]. Briefly, a filter paper saturated with 1 M NaOH was placed inside sealed wells containing transfected, differentiated H9c2 cells in fatty acid oxidation medium [12.5 mM HEPES, 0.3 % fatty acid-free BSA, 1 mM l-carnitine, 100 μM oleic acid medium containing 0.4 μCi/ml [1–¹⁴C]-oleate (PerkinElmer)] for 3 h at 37 °C. Disintegrations per minute from [1–¹⁴C]-CO₂ derived salts were measured in the filter paper to determine oleate oxidation using a TRI-CARB 5110 TR Liquid Scintillation Counter system (PerkinElmer). Results were normalized to total protein extracted from cells using RIPA buffer and standard protein extraction protocol.

Cells were pulsed overnight for 18 h with [1-¹⁴C] oleate (1 μCi/ml; PerkinElmer, Boston, MA) and cold oleate (100 μM) to prelabel the endogenous TAG pool. Oleate was coupled to FA-free BSA in a molar ratio of 5:1. Following the pulse, myotubes were chased for 3 h in DMEM containing 0.1 mM glucose, 0.5% FA-free BSA, and 10 μM triacsin C to block FA recycling into the TAG pool as described elsewhere44 (link), in absence or presence of 10 μM forskolin to stimulate lipolysis. For electrical pulse stimulation experiments, cells were chased for 24 h in DMEM containing 1 mM glucose, 0.5% FA-free BSA and 10 μM triacsin C while electrically stimulated by 2 ms pulses at a frequency of 0.1 Hz. TAG-derived FA oxidation was measured by the sum of ¹⁴CO₂ and ¹⁴C-ASM (acid soluble metabolites) in absence of triacsin C as previously described40 (link). Myotubes were harvested in 0.2 ml SDS 0.1% at the end of the pulse and of the chase period to determine oleate incorporation into TAG and protein content. The lipid extract was separated by TLC using heptane-isopropylether-acetic acid (60:40:4, v/v/v) as developing solvent. All assays were performed in duplicates, and data were normalized to cell protein content. Palmitate oxidation rate was measured as previously described43 (link).