INHIBITION OF ACYL-COA-CHOLESTEROL ACYLTRANSFERASE IN CHINESE-HAMSTEROVARY (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 shor t-chain N-acyl analogues. A potential role for ceramide in modulating cholesterol esterification was investigated using a series of short-ch ain ceramides and dihydroceramides. Acyl-CoA:cholesterol acyltransfera se (ACAT) in CHO cells was inhibited rapidly (< 30 min) and in a dose- dependent fashion by two N-acyl analogues of naturally occurring D-ery thro-ceramide, N-acetyl-sphingosine (D-erythro-C-2-ceramide) and N-hex anoyl-sphingosine (D-erythro-C-6-ceramide). At 10 mu M D-erythro-C-2-c eramide, esterification of cholesterol was inhibited by 95% in CHO cel ls grown in delipidated serum, and 80-85% in cells grown in 25-hydroxy cholesterol or human low-density lipoprotein (LDL). D-erythro-C-2-Cera mide did not inhibit [C-14]oleate-labelling of triacylglycerol and pho spholipid. Inhibition of cholesterol esterification in cells and isola ted membranes required the D-erythro (2S,3R) configuration (the L-thre o isomer of C-2-ceramide was not inhibitory) and an N-acyl group (sphi ngosine and sphinganine did not inhibit). DL-erythro-C-2-Dihydrocerami de was also a potent ACAT inhibitor in isolated membranes (IC50 0.2 mu M) and cells indicating lack of requirement for a 4-trans double bond . Consistent with results for C-2-ceramides, DL-threo-C-2-dihydroceram ide 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-C-2-ceramide, D-erythro-C-6- and C-4-ceramide w ere slightly weaker inhibitors of ACAT in isolated membranes. Thus, N- acyl chain length could influence inhibition, either by altering the e ffective concentration of ceramide in membranes or affinity for the AC AT enzyme. Short-chain ceramides and dihydroceramides are the first AC AT inhibitors described with structural similarity to a naturally occu rring compound.