Metabolism of short-chain ceramide and dihydroceramide analogues in Chinese hamster ovary (CHO) cells

A series of radiolabelled ceramides (d-erythro and l-threo) and dihydroceramides (dl-erythro and dl-threo) with 2, 4 or 6 carbon N-acyl groups were synthesized. These analogues were incubated with cultured CHO cells and radioactive products isolated and analyzed. In addition to synthesis of short-chain sphingomyelin and glucosylceramide, radiolabelled sphingosine and sphinganine were released from short-chain ceramides and dihydroceramides and subsequently utilized for synthesis of long-chain ceramide and sphingolipids. Substrate preference for short-chain sphingomyelin synthesis in cells was d-erythro-ceramides > l-threo-ceramides > dl-erythro-dihydroceramides > dl-threo-dihydroceramides, and C₄-and C₆-analogues were preferred over the C₂-analogue. Kinetic constants for conversion of short-chain (dihydro)ceramides to short-chain sphingomyelin were determined using CHO cell membranes and found to correlate with substrate preference in cultured cells. d-erythro-C₆-Ceramide was the preferred substrate for short-chain glucosylceramide synthesis. d-erythro-C₂-ceramide inhibited incorporation of [³H]serine into sphingomyelin, glucosylceramide and ceramide rapidly (2 h) and in a dose-dependent manner. Over a similar time period, [³H]choline-labelling of sphingomyelin was not affected. Inhibition of [³H]serine-labelling of sphingolipids appeared to correlate with release of [³H]long-chain bases from short-chain ceramides and dihydroceramides and synthesis of long-chain sphingolipids. However, some discrepancies between dl-erythro-C₄- and C₆-dihydroceramides, and d-erythro-C₂-ceramide suggested that short-chain dihydroceramides were less efficient in suppressing de novo synthesis from [³H]serine, while contributing substantially to endogenous sphingolipid synthesis. Inhibition of de novo sphingolipid synthesis by short-chain ceramides and dihydroceramides could not be related to inhibition of serine palmitoyltransferase activity in vitro.