Gas chromatography
Muscle samples were weighed, and disrupted with TissueLyser twice at 30 Hz for 3 min each using stainless steel beads and racks precooled at −80°C. After removing the beads, 1 mL ice-cold Ripa buffer was added per 100 mg of disrupted tissue, and samples were centrifuged at 2000 g for 10 min at 4°C. Two mg of proteins, according to the bicinchoninic acid assay in the supernatants, was mixed with 2.5 mL chloroform/methanol (1∶1). After vortex and sonication, samples were incubated for at least 2–3 hours at 4°C, and centrifuged at 2000 g for 10 min at 4°C. Supernatants were collected into glass tubes with a Pasteur pipette, and dried under nitrogen. Afterwards, the non-polar lipid fraction was obtained by separation on Sephadex columns. In the case of blood samples, they were collected on heparinized tubes by heart puncture from anesthetized mice, placed on ice, and centrifuged at 4000 rpm for 4 min at 4°C. Total lipids were extracted from plasma following a modified version of the Bligh and Dyer method [18]. Briefly, methanol/chloroform solution was added to plasma. Centrifugations were performed after addition of chloroform and KCl. Lower phases were collected and mixed with methanol. After a second centrifugation, the lower phase containing the lipids was collected on glass tubes. Extracted lipids from muscle and plasma were transmethylated by adding a mix of methanol and KOH. After collecting the samples in heptane, the so-generated fatty acid methyl esters were submitted to gas chromatography by using a Varian 3400 CX chromatograph fitted with a WCOT fused silica capillary column of 100 m×0.25 mm×0.20 µm (coated with polar highly substituted cyanopropyl CP-SIL 88 phase). The injection volume was 1 µL, and the split ratio was set at 1∶1. The temperature gradient in the oven ranged from 80 to 220°C at a rate of 4°C/min, and helium was the gas carrier. The temperature of the flame ionization detector was set at 270°C. Peaks were identified by retention time and compared to a standard mix of fatty acid methyl esters (Supelco 37 and Supelco PUFA-2 Animal Source; Sigma-Aldrich, Saint-Quentin Fallavier, France). Data were expressed as relative percentages.