Lipid metabolic changes caused by short-chain ceramides and the connection with apoptosis

The effects of the short-chain ceramides D-erythro-N-acetylsphingosine (C-2-ceramide), 6-[N-(7-nitrobenz-2-oxa-1,3-diazole-4-yl)amino]hexanoyl-D-erythro-sphingosine (NBD-ceramide) and N-[4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl]-D-erythro-sphingosine (DMB-ceramide) on the incorporation of [C-14]acetate into baby-hamster kidney (BHK) fibroblasts have been examined. C-2-ceramide at concentrations up to 20 mu M caused an inhibition of synthesis of phosphatidylcholine (PtdCho), sphingolipids and cholesterol within 2 h. Similar effects in BHK cells were seen using other radioactive tracers ([H-3]water, [H-3]palmitate and [H-3]choline) and using HL60 cells labelled with [C-14]acetate. The inhibition of PtdCho synthesis corresponded to an accumulation of label in diacylglycerol and triacylglycerol, probably as a consequence of cytidylyltransferase blockade. With [H-3]choline label, the decrease in sphingomyelin synthesis could be partly accounted for by accumulation of a slow-moving lipid, likely to be C-2-sphingomyelin. NBD-ceramide also reduced sphingomyelin and cholesterol biosynthesis, but had much less effect on PtdCho and acylglycerols. In contrast, the only apparent effect of DMB-ceramide was to inhibit synthesis of sphingomyelin, with a reciprocal increase in DMB-sphingomyelin synthesis. However, all of these short-chain ceramides caused massive apoptosis after 18 h, whereas addition of N-acetyldihydrosphingosine or elevation of natural ceramide by treatment of cells with sphingomyelinase had little effect on lipid synthesis or apoptosis. The present findings suggest that the apoptotic effect of short-chain ceramides is sometimes associated with inhibition of cytidylyltransferase, but is more closely correlated with a competitive inhibition of normal sphingomyelin biosynthesis.