Supporting Material
Material and Methods
Free fatty acids - Sample preparation and derivatization, GC-EI/MS and GC-NICI/MS
Free fatty acids - Sample preparation and derivatization. About 50mg mouse liver was
homogenized in 0.5ml of phosphate buffered saline and 0.5ml of methanol. For serum free
fatty acid determination 50µl serum were used. Each sample was immediately spiked with
1.2mol of a C15:0 fatty acid as internal standard. Following that, lipids were extracted
according to Bligh and Dyer. Lipid extracts were taken to dryness and redissolved in 50µl of
a pentafluoro benzyl bromide solution (3.4 % in acetonitrile) and 10µl of N,N-diisopropyl
ethanolamine. After 10min of incubation at room temperature samples were evaporated
under a gentle stream of nitrogen and resuspended in 50µl of hexane.
GC-EI/MS. A Trace-DSQ GC-MS (Thermo Scientific, Austin, Texas) equipped with a TRFAME 30m column was used. Helium was used as carrier gas at 1.3ml/min in split mode at
250°C injector temperature. The initial oven temperature of 150°C was held for 0.5min and
then the temperature was ramped up to 180°C at a rate of 10°C/min. This was followed by a
ramp of 0.5°C/min up to 190°C and 40°C/min up to 250°C where the temperature was held
for another 3min. The mass spectrometer was run in electron impact (EI) mode where the
FAs were detected in full scan of m/z 80 - 400. The source temperature was set to 250°C
and the transfer line temperature was 200°C. Peak areas for FAs were calculated by Xcalibur
QuanBrowser and related to the internal standard peak area.
GC-NICI/MS. A Trace-DSQ GC-MS (Thermo Scientific) equipped with a TR-FAME 30m
column was used in splittless mode with 1ml/min helium as carrier gas and 300°C injector
temperature. The initial oven temperature of 150°C was held for 1min and then the
temperature first was ramped up to 200°C at a rate of 25°C/min. This was followed by a ramp
of 12.5°C/min up to 325°C where the temperature was held for another 2min. The mass
spectrometer was run in negative ion chemical ionization (NICI) mode where the fatty acids
were detected in full scan as carboxylates after loss of the pentafluoro benzyl moiety.
Methane was used as CI gas, the source temperature was set to 250°C and the transfer line
temperature was 330°C.
Cell culture experiments.
DMEM was supplemented with 10% fetal calf serum, 1% streptomycin/penicillin, sodium
pyruvate, glutamine and non-essential amino acids at 37°C in a humidified 5% CO2
atmosphere. Plasmid shRNA and lentivirus packaging mix for ATGL knockdown were from
Sigma Aldrich and used according to the manufacturer’s instructions. The medium was
changed every 48 hours.
For palmitic acid (PA) or oleic acid (OA) treatment Hepa1.6 cells were precultured overnight
in DMEM without fetal calf serum and treated with oleate 500µM, palmitate 500µM and TM
1µg/ml and harvested at the indicated time points. Experiments were repeated in triplicates.
5mM stock solutions of OA and PA were prepared by dissolving the substances in a mixture
of 0.01M NaOH (25%) and 150mM NaCl (40%) while heating. After heating a 24% FA free
BSA solution (in 150mM NaCl) was added (18%). Finally PBS (10%) was added and filled up
with distilled sterile water to 100%.