title: Determining the Turnover of Glycosphingolipid Species by Stable-Isotope Tracer Lipidomics.
abstract: In this study, we used water-soluble stable mass isotope precursors to measure the turnover of endogenous ceramide (Cer) and glycosphingolipids (GSLs) in HEp-2 cells. Cells incubated in the presence of [13C-U]glucose showed rapid incorporation of hexose residues with an increased mass of 6Da into GSLs. Different turnover rates of GSL classes and their molecular species were observed. Approximately 30% of the glucosylceramide, 50% of the lactosylceramide, and 50% of the globotriaosylceramide species showed a much slower turnover than the rest. This demonstrates the existence of different lipid pools, where a certain fraction of species survived for a long time in the cells. The species with the shortest N-amidated fatty acyl groups (C16:0 and C18:0) showed a more rapid turnover than those with the longest N-amidated fatty acids (C24:0 and C24:1). Experiments with addition of [13C-U]serine were performed to study de novo synthesis of Cer from serine and palmitoyl-CoA. These experiments revealed that de novo synthesis contributes to a minor extent to the total synthesis of new sphingolipids and showed that there is a more rapid formation of the longest Cer species (C24:0 and C24:1) than of the shortest species (C16:0), that is, the opposite as observed for the GSLs in the experiments with [13C-U]glucose. In conclusion, this FLUX lipidomics experimental approach with the addition of [13C-U]glucose to cells allows us to not only study the total turnover but also permit observations of lipid intermediates and metabolic flow of endogenous GSL species at the molecular lipid level.
authors.affiliation: Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital-The Norwegian Radium Hospital, 0379 Oslo, Norway; Centre for Cancer Biomedicine, Faculty of Medicine, University of Oslo, 0379 Oslo, Norway. Electronic address: Tore.Skotland@rr-research.no.
authors.forename: Tore
authors.initials: T
authors.lastname: Skotland
authors.affiliation: Zora Biosciences Oy, 02150 Espoo, Finland. Electronic address: kekroos@hotmail.com.
authors.forename: Kim
authors.initials: K
authors.lastname: Ekroos
authors.affiliation: Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital-The Norwegian Radium Hospital, 0379 Oslo, Norway; Centre for Cancer Biomedicine, Faculty of Medicine, University of Oslo, 0379 Oslo, Norway; Department of Molecular Biosciences, University of Oslo, 0316 Oslo, Norway.
authors.forename: Simona
authors.initials: S
authors.lastname: Kavaliauskiene
authors.affiliation: Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital-The Norwegian Radium Hospital, 0379 Oslo, Norway; Centre for Cancer Biomedicine, Faculty of Medicine, University of Oslo, 0379 Oslo, Norway.
authors.forename: Jonas
authors.initials: J
authors.lastname: Bergan
authors.affiliation: Zora Biosciences Oy, 02150 Espoo, Finland.
authors.forename: Dimple
authors.initials: D
authors.lastname: Kauhanen
authors.affiliation: Zora Biosciences Oy, 02150 Espoo, Finland.
authors.forename: Tuulia
authors.initials: T
authors.lastname: Lintonen
authors.affiliation: Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital-The Norwegian Radium Hospital, 0379 Oslo, Norway; Centre for Cancer Biomedicine, Faculty of Medicine, University of Oslo, 0379 Oslo, Norway; Department of Molecular Biosciences, University of Oslo, 0316 Oslo, Norway.
authors.forename: Kirsten
authors.initials: K
authors.lastname: Sandvig
mesh_terms: D002247:Carbon Isotopes / Q000378:metabolism; D002460:Cell Line; D004847:Epithelial Cells / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D006028:Glycosphingolipids / Q000378:metabolism*; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D007554:Isotopes / Q000378:metabolism*; D012694:Serine / Q000378:metabolism
chemical_list: D002247:Carbon Isotopes; D006028:Glycosphingolipids; D007554:Isotopes; D012694:Serine; D005947:Glucose
keywords: FLUX lipidomics; lipid turnover; mass isotopes; mass spectrometry; sphingolipids