Sphingolipid Uptake by Cultured Cells

Abstract

Human fibroblasts, rat neurons, and murine neuroblastoma cells, cultured in the presence of fetal calf serum, were fed with [1-3 H]sphingosine to radiolabel sphingolipids. The fate of cell sphingolipids, the release of sphingolipids in the culture medium, the interaction of sphingolipids with the proteins and lipoproteins of fetal calf serum, and the fate of sphingolipids taken up by the cells were investigated. For this latter purpose, the culture medium containing radioactive sphingolipids was delivered to nonlabeled cells. The presence of tritium at position 1 of sphingosine allowed us to follow the extent of sphingolipid catabolism by measuring the production of radioactive phosphatidylethanolamine and proteins by recycling the radioactive ethanolamine formed during sphingosine catabolism and the production of tritiated water. We confirmed that in cells the recycling of sphingosine occurred to a high extent and that only a minor portion of cell sphingolipids was catabolized to the small fragments of ethanolamine and water. Cell sphingolipids were released in the culture medium, where they formed large lipoproteic aggregates at a rate of about 12% per day. Released sphingolipids were taken up by the cells and catabolized to the sphingosine and then to ethanolamine, and recycling of sphingosine was not observed. This suggests that in the presence of fetal calf serum in the culture medium, exogenous sphingolipids directly reach the lysosomes, were they are entirely catabolized. Thus, the trafficking of sphingolipids from cells to the extracellular environment and from this to other cells does not allow the modification of the plasma membrane composition.Membrane sphingolipids are complex lipids displaying a strong amphiphilic character (1). This implies an equilibrium between plasma membrane molecules and free monomers present in the extracellular environment. The critical aggregative concentration of sphingolipids is in the nano-or picomolar range (1, 2), thus the amount of free monomers in solution is low. Nevertheless, the quantity of free monomers should increase when a favorable equilibrium leads to the formation of stable lipoprotein complexes (3-5). This process should also occur in living organisms, where significant amounts of sphingolipids are detectable in serum and cerebrospinal fluid from normal and pathological subjects (6 -8), as components of serum lipoproteins or associated with albumin (4).The presence of sphingolipids in biological fluids leads to the following considerations. Exogenous sphingolipids administered to cells are taken up by the cells, thus entering the sphingolipid metabolic pathway. Several studies have been carried out by administering sphingolipids to cells (9). Gangliosides administered to cells that are cultured in the absence of fetal calf serum (FCS) 1 or proteins have been shown to become components of the cell membranes, modifying in some cases the quantity and the quality of plasma membrane gangliosides, and to enter into the endogenous sphingolipid metabolic pathways ...

Differential Effects of Ceramide and Sphingosine 1-Phosphate on ERM Phosphorylation

“…The effects of bSMase on ERM proteins reported previously by our group (20) could be exerted by the effect of bSMase on cellular SM or on the SM associated with lipoproteins found in FBS. We observed that FBS used in cell culture is rich in SM, consistent with previous studies (35). Thus, bSMase treatment of FBS medium caused hydrolysis of SM not only in the cells but also in the medium.…”

Alternation in the Glycolipid Transfer Protein Expression Causes Changes in the Cellular Lipidome

“…The acyl chains of ether-linked lipids and the N-linked acyl chains for sphingomyelin (SM), ceramide (Cer) and glycosphingolipids (GSLs) are shown after the slash. All samples, including the controls were cultured in the presence of the same amount of media and FCS and we assume that the small amount of FCS in the culture media is not affecting the cellular lipid metabolism [18].…”

Sphingolipid and Glycosphingolipid Metabolic Pathways in the Era of Sphingolipidomics

“…(484) Hydrolytic enzymes for sphingolipids are also found in other locations in the cell to produce bioactive products for cell signaling 5,6 and rearrangement of membrane architecture. 23,485 The complete balance sheet for sphingolipids additionally includes uptake of sphingolipids from exogenous sources (such as albumin and lipoproteins) 486,487 and losses by efflux,(488) lipoprotein secretion, 354,489 and shedding of membrane vesicles containing sphingolipids. (490)…”