Metabolites were extracted from serum samples (thawed on ice) by adding 65 μl 80:20 methanol:water solution at −80 °C to 5 μl of serum sample, followed by vortexing for 10 s, incubation at 4 °C for 10 min, and centrifugation at 4 °C and 16,000g· for 10 min. To extract metabolites from tissue samples, frozen tissue samples were first weighed (~20 mg each sample) and ground using a Cryomill (Retsch). The resulting powder was then mixed with −20 °C 40:40:20 methanol:acetonitrile:water solution, followed by vortexing for 10 s, incubation at 4 °C for 10 min, and centrifugation at 4 °C and 16,000g for 10 min. The volume of the extraction solution (in μl) was 40 × the weight of tissue (in mg). The supernatant was transferred to LC–MS autosampler vials for analysis.
Serum and tissue extracts were analysed using LC–MS. In brief, a quadrupoleorbitrap mass spectrometer (Q Exactive Plus, Thermo Fisher Scientific) operating in negative ion mode was coupled to hydrophilic interaction chromatography via electrospray ionization and used to scan from m/z 73 to 1,000 at 1 Hz and 140,000 resolution. LC separation was achieved on a XBridge BEH Amide column (2.1 mm × 150 mm, 2.5 μm particle size, 130 Å pore size; Waters) using a gradient of solvent A (20 mM ammonium acetate + 20mM ammonium hydroxide in 95:5 water:acetonitrile, pH 9.45) and solvent B (acetonitrile). Flow rate was 150 μl min−1. The gradient was: 0 min, 85% B; 2 min, 85% B; 3 min, 80% B; 5 min, 80% B; 6 min, 75% B; 7 min, 75% B; 8 min, 70% B; 9 min, 70% B; 10 min, 50% B; 12 min, 50% B; 13 min, 25% B; 16 min, 25% B; 18 min, 0% B; 23 min, 0% B; 24 min, 85% B; 30 min, 85% B. Data were analysed using the MAVEN software35 (link). Isotope labelling was corrected for natural abundances of 13C, 2H, and 15N. Circulating glycerol labelling was determined by first converting serum glycerol to glycerol-3-phosphate using glycerol kinase and then measuring the labelling of glycerol-3-phosphate with LC–MS.
For acetate, circulating metabolite labelling was determined by GC–MS using a 7890A GC system coupled to a 5975 MSD mass spectrometer (Agilent) after derivatization with 2,3,4,5,6-pentafluorobenzyl bromide as described36 (link). GC separation was achieved using an Agilent J&W 122-7033 column (30 m × 0.25 mm × 0.5 μm). The GC temperature program was: 0 min, 35 °C; 6 min, 35 °C; 12 min, 220 °C; 17 min, 220 °C, followed by returning to 35 °C for the next injection. Other GC parameters were: injection volume 1 μl; He as carrier gas at a flow rate of 1.2 ml min−1; inlet temperature 250 °C; transfer line temperature 280 °C. Mass spectrometry detection was in electron impact ionization mode, with SIM scans of m/z 240.3 and 242.3 for unlabelled and 13C2-acetate, respectively.
Free fatty acids in serum samples (thawed on ice) were extracted by adding 200 μl ethyl acetate at room temperature to 10 μl serum samples, followed by vortexing for 10 s, incubation at 4 °C for 10 min, and centrifugation at 16,000g for 10 min. The top layer of approximately 190 μl was transferred to a new glass vial before being dried under nitrogen gas flow. The dried extract was dissolved in 100 μl 1:1 isopopanol: methanol before being loaded onto the LC–MS. MS analysis was conducted on an Exactive orbitrap mass spectrometer (Thermo Fisher Scientific) scanning at 1 Hz and 100,000 resolution operating in negative ion mode. LC separation was on reversed-phase ion-pairing chromatography on a Luna C8 column (150 × 2.0 mm, 3 μm particle size, 100 Å pore size; Phenomenex) with a gradient of solvent A (10 mM tributylamine + 15 mM acetic acid in 97:3 water:methanol, pH 4.5) and solvent B (methanol). Flow rate was 250 μl min−1. The gradient was: 0 min, 80% B; 10 min, 90% B; 11 min, 99% B; 25 min, 99% B; 26 min, 80% B; 30 min, 80% B.
Serum and tissue extracts were analysed using LC–MS. In brief, a quadrupoleorbitrap mass spectrometer (Q Exactive Plus, Thermo Fisher Scientific) operating in negative ion mode was coupled to hydrophilic interaction chromatography via electrospray ionization and used to scan from m/z 73 to 1,000 at 1 Hz and 140,000 resolution. LC separation was achieved on a XBridge BEH Amide column (2.1 mm × 150 mm, 2.5 μm particle size, 130 Å pore size; Waters) using a gradient of solvent A (20 mM ammonium acetate + 20mM ammonium hydroxide in 95:5 water:acetonitrile, pH 9.45) and solvent B (acetonitrile). Flow rate was 150 μl min−1. The gradient was: 0 min, 85% B; 2 min, 85% B; 3 min, 80% B; 5 min, 80% B; 6 min, 75% B; 7 min, 75% B; 8 min, 70% B; 9 min, 70% B; 10 min, 50% B; 12 min, 50% B; 13 min, 25% B; 16 min, 25% B; 18 min, 0% B; 23 min, 0% B; 24 min, 85% B; 30 min, 85% B. Data were analysed using the MAVEN software35 (link). Isotope labelling was corrected for natural abundances of 13C, 2H, and 15N. Circulating glycerol labelling was determined by first converting serum glycerol to glycerol-3-phosphate using glycerol kinase and then measuring the labelling of glycerol-3-phosphate with LC–MS.
For acetate, circulating metabolite labelling was determined by GC–MS using a 7890A GC system coupled to a 5975 MSD mass spectrometer (Agilent) after derivatization with 2,3,4,5,6-pentafluorobenzyl bromide as described36 (link). GC separation was achieved using an Agilent J&W 122-7033 column (30 m × 0.25 mm × 0.5 μm). The GC temperature program was: 0 min, 35 °C; 6 min, 35 °C; 12 min, 220 °C; 17 min, 220 °C, followed by returning to 35 °C for the next injection. Other GC parameters were: injection volume 1 μl; He as carrier gas at a flow rate of 1.2 ml min−1; inlet temperature 250 °C; transfer line temperature 280 °C. Mass spectrometry detection was in electron impact ionization mode, with SIM scans of m/z 240.3 and 242.3 for unlabelled and 13C2-acetate, respectively.
Free fatty acids in serum samples (thawed on ice) were extracted by adding 200 μl ethyl acetate at room temperature to 10 μl serum samples, followed by vortexing for 10 s, incubation at 4 °C for 10 min, and centrifugation at 16,000g for 10 min. The top layer of approximately 190 μl was transferred to a new glass vial before being dried under nitrogen gas flow. The dried extract was dissolved in 100 μl 1:1 isopopanol: methanol before being loaded onto the LC–MS. MS analysis was conducted on an Exactive orbitrap mass spectrometer (Thermo Fisher Scientific) scanning at 1 Hz and 100,000 resolution operating in negative ion mode. LC separation was on reversed-phase ion-pairing chromatography on a Luna C8 column (150 × 2.0 mm, 3 μm particle size, 100 Å pore size; Phenomenex) with a gradient of solvent A (10 mM tributylamine + 15 mM acetic acid in 97:3 water:methanol, pH 4.5) and solvent B (methanol). Flow rate was 250 μl min−1. The gradient was: 0 min, 80% B; 10 min, 90% B; 11 min, 99% B; 25 min, 99% B; 26 min, 80% B; 30 min, 80% B.
