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

Abstract

The effects of the short-chain ceramides D-erythro-N-acetylsphingosine (C2-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 [14C]acetate into baby-hamster kidney (BHK) fibroblasts have been examined. C2-ceramide at concentrations up to 20 μM 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 ([3H]water, [3H]palmitate and [3H]choline) and using HL60 cells labelled with [14C]acetate. The inhibition of PtdCho synthesis corresponded to an accumulation of label in diacylglycerol and triacylglycerol, probably as a consequence of cytidylyltransferase blockade. With [3H]choline label, the decrease in sphingomyelin synthesis could be partly accounted for by accumulation of a slow-moving lipid, likely to be C2-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.

Bioconjugation Strategies for Revealing the Roles of Lipids in Living Cells

“…Previous studies reported the apoptotic effect of permeable unnatural short-chain ceramides on cells. 30,31 However, short-chain ceramide analogues differ significantly in their physical membrane properties as well as biological activity compared to natural long-chain ceramides. 32,33 Therefore, these short-chain analogues are limited in their use in structure−function analyses relevant to natural ceramides and do not enable direct studies on how the structure of the N-acyl group of ceramides affects apoptosis.…”

“…Our initial experiments enabled us to directly detect the apoptotic effect of saturated long-chain ceramides within cells using in situ lipid synthesis, confirming previous conclusions that were made indirectly by using short-chain ceramides. 30 However, the effect of N-acyl chain length or saturation on ceramide function had not been directly investigated in previous studies. Human cells produce a number of different ceramide species that differ in N-acyl chain length.…”

“…They are formed by ceramide synthases that N-acylate long-chain sphingoid bases with various fatty acids. , Although known to be involved in signaling and programmed cell death, the exact roles and interactions of the various known ceramide species remain elusive. Previous studies reported the apoptotic effect of permeable unnatural short-chain ceramides on cells. , However, short-chain ceramide analogues differ significantly in their physical membrane properties as well as biological activity compared to natural long-chain ceramides. , Therefore, these short-chain analogues are limited in their use in structure–function analyses relevant to natural ceramides and do not enable direct studies on how the structure of the N-acyl group of ceramides affects apoptosis.…”