Inhibition of acyl-CoA:cholesterol acyltransferase in Chinese hamster ovary (CHO) cells by short-chain ceramide and dihydroceramide.
The biological activity of ceramide, an intermediate in the synthesis and catabolism of sphingolipids, has been shown to be mimicked by short-chain N-acyl analogues. A potential role for ceramide in modulating cholesterol esterification was investigated using a series of short-chain ceramides and dihydroceramides. Acyl-CoA:cholesterol acyltransferase (ACAT) in CHO cells was inhibited rapidly (< 30 min) and in a dose-dependent fashion by two N-acyl analogues of naturally occurring D-erythro-ceramide, N-acetyl-sphingosine (D-erythro-C2-ceramide) and N-hexanoyl-sphingosine (D-erythro-C6-ceramide). At 10 microM D-erythro-C2-ceramide, esterification of cholesterol was inhibited by 95% in CHO cells grown in delipidated serum, and 80-85% in cells grown in 25-hydroxycholesterol or human low-density lipoprotein (LDL). D-erythro-C2-Ceramide did not inhibit [14C]oleate-labelling of triacylglycerol and phospholipid. Inhibition of cholesterol esterification in cells and isolated membranes required the D-erythro (2S,3R) configuration (the L-threo isomer of C2-ceramide was not inhibitory) and an N-acyl group (sphingosine and sphinganine did not inhibit). DL-erythro-C2-Dihydroceramide was also a potent ACAT inhibitor in isolated membranes (IC50 0.2 microM) and cells indicating lack of requirement for a 4-trans double bond. Consistent with results for C2-ceramides, DL-threo-C2-dihydroceramide was not inhibitory in cells or in vitro. Long-chain ceramide and N-palmitoyl-dihydroceramide did not inhibit ACAT in isolated membranes. Compared to D-erythro-C2-ceramide, D-erythro-C6- and C4-ceramide were slightly weaker inhibitors of ACAT in isolated membranes. Thus, N-acyl chain length could influence inhibition, either by altering the effective concentration of ceramide in membranes or affinity for the ACAT enzyme. Short-chain ceramides and dihydroceramides are the first ACAT inhibitors described with structural similarity to a naturally occurring compound.
Ridgway
N D
ND
Department of Pediatrics, Dalhousie University, Halifax, Nova Scotia, Canada.
D000818:Animals; D016466:CHO Cells / Q000201:enzymology*; D002518:Ceramides / Q000494:pharmacology*; D002784:Cholesterol / Q000378:metabolism; D006224:Cricetinae; D004951:Esterification; D006888:Hydroxycholesterols / Q000494:pharmacology; D007700:Kinetics; D008077:Lipoproteins, LDL / Q000494:pharmacology; D013110:Sphingosine / Q000031:analogs & derivatives / Q000494:pharmacology; D002785:Sterol O-Acyltransferase / Q000037:antagonists & inhibitors*
D002518:Ceramides; D006888:Hydroxycholesterols; D008077:Lipoproteins, LDL; C064769:N-acetylsphingosine; C007997:25-hydroxycholesterol; D002784:Cholesterol; D002785:Sterol O-Acyltransferase; D013110:Sphingosine