Adipose tissue glucose uptake, fatty acid profile, and ceramide content

Adipose tissue insulin-stimulated glucose uptake and fatty acid profile were evaluated essentially as previously described (9, 26). For ceramide quantification, briefly, adipose tissue was homogenized in 10 mM sodium phosphate buffer (pH 7.4) containing 100 μM deferoxamine mesylate and the internal standard, N-heptadecanoyl-D-erythro-sphingosylphosphorylcholine (SM d18:1/17:0). Lipids were extracted with methyl-tert-butyl ether/methanol. The organic phase containing lipids was dried under nitrogen stream, suspended in isopropanol, and analyzed in an ultra (U)HPLC (Nexera; Shimadzu, Kyoto, Japan) coupled to an electrospray ionization TOF mass spectrometer (Triple TOF 6600; Sciex, Framingham, MA). Lipids were separated on a UPLC reversed-phase column (CORTECS® C18 column, 1.6 μm, 2.1 mm internal diameter × 100 mm). Lipid molecular species were identified with an in-house Excel-based macro based on the MS and MS/MS fragmentation pattern observed with PeakView®. Quantification was performed with MultiQuant®, where peak areas of precursor ions were normalized to those of the internal standards.