Lipidomics analysis in subcutaneous white adipose tissue from adipose-selective FASN-KO, ACC1-KO and MCD-KO and their respective flex/flox controls mice
Description
Lipidomic analysis in inguinal white adipose tissue (iWAT) from adipocyte selective FASN, ACC1 and MCD KO mice and their respective controls. The results indicate the reduction of free palmitate by FASN and ACC1 deletion, but not MCD inactivation in adipocytes. Both FASN and ACC1 are key enzymes in de novo fatty acid biosynthesis in adipocytes. In addition, mass spectrometry analyses indicated the levels of acyl-CoAs and acyl-carnitines in iWAT from cAdFASN-KO, cAdACC1-KO, cAdMCD-KO and control mice.
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The indicated lipid species were analyzed using multidimensional mass spectrometry-based shotgun lipidomic analysis. To this end, adipose tissue homogenate containing 0.5 mg of protein (Pierce BCA assay) was accurately transferred to a disposable glass culture test tube. A premixture of lipid internal standards (IS) was added prior to conducting lipid extraction for quantification of the targeted lipid species. Lipid extraction was performed using a modified Bligh and Dyer procedure, and each lipid extract was reconstituted in chloroform:methanol (1:1, v:v) at a volume of 400 µL/mg protein. For shotgun lipidomics, lipid extract was further diluted to a final concentration of ~500 fmol total lipids per µL. Mass spectrometric analysis was performed on a triple quadrupole mass spectrometer (TSQ Altis, Thermo Fisher Scientific, San Jose, CA), which was equipped with an automated nanospray device (TriVersa NanoMate, Advion Bioscience Ltd., Ithaca, NY)87. Identification and quantification of lipid species were performed using an automated software program. Data processing (e.g., ion peak selection, baseline correction, data transfer, peak intensity comparison and quantitation) was performed and result was normalized to the protein content (nmol lipid/mg protein) or wet tissue weight (nmol lipid/mg tissue).