Abstract
Ceramides can be delivered to cultured cells without solvents in the form of complexes with cholesteryl phosphocholine. We have analysed the delivery of three different radiolabeled D-erythro-ceramides (C6-Cer, C10-Cer and C16-Cer) to HeLa cells, and followed their metabolism as well as the cell viability. We found that all three ceramides were successfully taken up by HeLa cells when complexed to CholPC in an equimolar ratio, and show that the ceramides show different rates of cellular uptake and metabolic fate. The C6-Cer had the highest incorporation rate, followed by C10-Cer and C16-Cer, respectively. The subsequent effect on cell viability strongly correlated with the rate of incorporation, where C6-Cer had the strongest apoptotic effects. Low-dose (1 μM) treatment with C6-Cer favoured conversion of the precursor to sphingomyelin, whereas higher concentrations (25–100 μM) yielded increased conversion to C6-glucosylceramide. Similar results were obtained for C10-Cer. In the lower-dose C16-Cer experiments, most of the precursor was degraded, whereas at high-dose concentrations the precursor remained un-metabolized. Using this method, we demonstrate that ceramides with different chain lengths clearly exhibit varying rates of cellular uptake. The cellular fate of the externally delivered ceramides are clearly connected to their rate of incorporation and their subsequent effects on cell viability may be in part determined by their chain length.
Highlights
Sphingolipids are a major class of lipids that are ubiquitously present in the membranes of eukaryotes, where they function as important structural components and exhibit important roles as bioactive molecules
To measure the cellular uptake of the different ceramides, HeLa cells were subjected to 50 μM of the various chain length, [3H]sphingosine labeled ceramides, complexed with cholesteryl phosphocholine (CholPC) in a 1:1 molar ratio
We demonstrate that ceramides with different chain lengths clearly exhibit varying rates of cellular uptake
Summary
Sphingolipids are a major class of lipids that are ubiquitously present in the membranes of eukaryotes, where they function as important structural components and exhibit important roles as bioactive molecules. Ceramide (Cer) has emerged as a central molecule in the sphingolipid metabolism, by serving as a precursor for all complex sphingolipids and displaying its involvement in a variety of cellular processes, including cell growth, signalling, proliferation, differentiation and apoptosis [1,2,3,4,5]. De novo synthesis of ceramide begins at the endoplasmatic reticulum, with a process that yields the long-chain amino-alcohol base, dihydrosphingosine. The dihydrosphingosine base is further modified by the addition of an acyl group by a ceramide synthase, yielding dihydroceramide. Introduction of a trans double bond between carbons 4 and 5 on the amino-alcohol base gives rise to ceramide [6,7,8].
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