BACKGROUND: Sphingolipids, e.g. ceramide (Cer), glucosylceramide (GlcCer) and sphingomyelin (SM), are important bulk constituents of plasma membranes in mammalian cells. In addition, these lipids are also enriched in certain intracellular organelles, as well as in the epidermal lamellar bodies (LBs) of differentiating keratinocytes (KCs). Epidermal Cer, which comprises a heterogeneous family of at least 10 members, is a key component of the stratum corneum (SC) lipids, and regulates permeability barrier function. Levels of GlcCer, but not SM, significantly increase during epidermal differentiation, and then both GlcCer and SM are enzymatically hydrolysed to Cer at and just above the transition from the stratum granulosum to the SC. OBJECTIVES: To determine: (i) whether the GlcCer contained in different pools, i.e. the membrane fraction or the LB fraction, has different metabolic fates; and (ii) whether specific molecular species of GlcCer localize to distinct subcellular pools. METHODS: To study the metabolic fate of specific molecular fractions of GlcCer and Cer, we first ascertained the full spectrum of molecular species present in cultured normal human KCs (CHK) in a differentiated condition as shown in vivo in epidermis. Cer species were analysed in CHK using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Next, the metabolic fate of the GlcCer was studied by pulse-labelling of CHK with L-[14C]-serine. RESULTS: The GlcCer of undifferentiated KCs comprised GlcCer B and Cer NS (or Cer 2), which contain nonhydroxy fatty acid (FA) as the amide-linked FA, while differentiated KCs displayed further heterogeneity of both GlcCer and Cer, including the presence of acylGlcCer and acylCer. The metabolic fates of these sphingolipids were determined. The GlcCer B level decreased over 2 days and then plateaued between days 3 and 5 following pulse-labelling of sphingolipids for 24 h. As GlcCer B declined, Cer NS (Cer 2) increased in a similar time-dependent manner. In contrast, both acylGlcCer and acylCer increased continuously in parallel over this experimental period. CONCLUSIONS: Distinct GlcCer pools segregated to those that were either hydrolysable or nonhydrolysable in differentiated KCs. We assume that the latter pool appears to be LB enriched, and also sequestrates acylGlcCer from other cellular membrane fractions.
BACKGROUND:Sphingolipids, e.g. ceramide (Cer), glucosylceramide (GlcCer) and sphingomyelin (SM), are important bulk constituents of plasma membranes in mammalian cells. In addition, these lipids are also enriched in certain intracellular organelles, as well as in the epidermal lamellar bodies (LBs) of differentiating keratinocytes (KCs). Epidermal Cer, which comprises a heterogeneous family of at least 10 members, is a key component of the stratum corneum (SC) lipids, and regulates permeability barrier function. Levels of GlcCer, but not SM, significantly increase during epidermal differentiation, and then both GlcCer and SM are enzymatically hydrolysed to Cer at and just above the transition from the stratum granulosum to the SC. OBJECTIVES: To determine: (i) whether the GlcCer contained in different pools, i.e. the membrane fraction or the LB fraction, has different metabolic fates; and (ii) whether specific molecular species of GlcCer localize to distinct subcellular pools. METHODS: To study the metabolic fate of specific molecular fractions of GlcCer and Cer, we first ascertained the full spectrum of molecular species present in cultured normal human KCs (CHK) in a differentiated condition as shown in vivo in epidermis. Cer species were analysed in CHK using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Next, the metabolic fate of the GlcCer was studied by pulse-labelling of CHK with L-[14C]-serine. RESULTS: The GlcCer of undifferentiated KCs comprised GlcCer B and Cer NS (or Cer 2), which contain nonhydroxy fatty acid (FA) as the amide-linked FA, while differentiated KCs displayed further heterogeneity of both GlcCer and Cer, including the presence of acylGlcCer and acylCer. The metabolic fates of these sphingolipids were determined. The GlcCer B level decreased over 2 days and then plateaued between days 3 and 5 following pulse-labelling of sphingolipids for 24 h. As GlcCer B declined, Cer NS (Cer 2) increased in a similar time-dependent manner. In contrast, both acylGlcCer and acylCer increased continuously in parallel over this experimental period. CONCLUSIONS: Distinct GlcCer pools segregated to those that were either hydrolysable or nonhydrolysable in differentiated KCs. We assume that the latter pool appears to be LB enriched, and also sequestrates acylGlcCer from other cellular membrane fractions.