Inhibition of acyl-CoA: cholesterol acyltransferase in chinese hamster ovary (CHO) cells by short-chain ceramide and dihydroceramide

Abstract

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-C 2-ceramide) and N-hexanoyl-sphingosine ( d- erythro-C 6-ceramide). At 10 μM d- erythro-C 2-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-C 2-Ceramide did not inhibit [ 14C]oleate-labelling of triacylglycerol and phospholipid. Inhibition of cholesterol esterification in cells and isolated membranes required the d- erythro (2 S,3 R) configuration (the l- threo isomer of C 2-ceramide was not inhibitory) and an N-acyl group (sphingosine and sphinganine did not inhibit). dl- erythro-C 2-Dihydroceramide was also a potent ACAT inhibitor in isolated membranes (IC 50, 0.2 μM) and cells indicating lack of requirement for a 4- trans double bond. Consistent with results for C 2-ceramides, dl- threo-C 2-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-C 2-ceramide, d- erythro-C 6- and C 4-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.