Ceramide Drives Cholesterol Out of the Ordered Lipid Bilayer Phase into the Crystal Phase in 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/Cholesterol/Ceramide Ternary Mixtures

Abstract

The effect of brain ceramide on the maximum solubility of cholesterol in ternary mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), cholesterol, and ceramide was investigated at 37 degrees C by a cholesterol oxidase (COD) reaction rate assay and by optical microscopy. The COD reaction rate assay showed a sharp increase in cholesterol chemical potential as the cholesterol mole fraction approaches the solubility limit. A decline in the COD reaction rate was found after the formation of cholesterol crystals. The maximum solubility of brain ceramide in POPC bilayers was determined to be 68 +/- 2 mol % by microscopy. We found that ceramide has a much higher affinity for the ordered bilayers than cholesterol, and the maximum solubility of cholesterol decreases with the increase in ceramide content. More significantly, the displacement of cholesterol by ceramide follows a 1:1 relation. At the cholesterol solubility limit, adding one more ceramide molecule to the lipid bilayer drives one cholesterol out of the bilayer into the cholesterol crystal phase, and cholesterol is incapable of displacing ceramide from the bilayer phase. On the basis of these findings, a ternary phase diagram of the POPC/cholesterol/ceramide mixture was constructed. The behaviors of ceramide and cholesterol can be explained by the umbrella model. Both ceramide and cholesterol have small polar headgroups and relatively large nonpolar bodies. In a PC bilayer, ceramide and cholesterol compete for the coverage of the headgroups of neighboring PC to prevent the exposure of their nonpolar bodies to water. This competition results in the 1:1 displacement as well as the displacement of cholesterol by ceramide from lipid raft domains.

Enzymatic generation of ceramide induces membrane restructuring: Correlated AFM and fluorescence imaging of supported bilayers

“…These conclusions are consistent with reports that ceramide incorporation leads to displacement of cholesterol from ordered domains in both model membranes and cells (Ali et al, 2006;Chiantia et al, 2006Chiantia et al, , 2007Megha et al, 2006;Megha and London, 2004;Yu et al, 2005b) and with the reported ability of ceramide to substitute for cholesterol in liquid-ordered phases (Carrer et al, 2006;Silva et al, 2007). However, the correlated imaging results suggest that the membrane restructuring initiated by SMase may be more complex than simple loss of cholesterol from a ceramide-rich domain.…”

“…This work has demonstrated that ceramide leads to increased membrane heterogeneity and that its effects depend on the membrane composition. Ceramide has also been shown to displace cholesterol from raft domains in model and cellular membranes (Ali et al, 2006;Chiantia et al, 2006;Megha and London, 2004;Megha et al, 2006;Yu et al, 2005b). Several recent studies have concluded that the ability of ceramide to modulate the properties of liquid-ordered domains and form ceramide-enriched gel domains is affected by the chain length and that short-chain ceramides (<C 12 ) are not good models for studies of the biological activity of ceramide (Chiantia et al, 2007;Megha et al, 2007).…”

Cholesterol homeostasis and the escape tendency (activity) of plasma membrane cholesterol

“…Further support for this hypothesis is the evidence that various ceramides and diglycerides not only displace cholesterol from liquid-ordered systems but also can substitute for the sterol [95,101]. Similarly, ceramide and diglyceride analogues, as well as 1-octanol, both substitute for red cell membrane cholesterol and displace it from phospholipids, as indicated by their ability to increase its sensitivity to cholesterol oxidase and the rate of its transfer to cyclodextrin [54,81].…”

“…Surprisingly, amphipaths as disparate as 1-octanol and short-chain ceramide and diglyceride analogues also prevent lysis. Indeed, ceramides have a higher affinity than cholesterol for ordered phases of 1-palmitoyl-2-oleoyl-phosphatidylcholine [95]. It also appears that ceramides can form condensed complexes with phospholipids like sphingomyelin [96,97].…”

Generating Ceramide from Sphingomyelin by Alkaline Sphingomyelinase in the Gut Enhances Sphingomyelin-Induced Inhibition of Cholesterol Uptake in Caco-2 Cells

“…It might be related with the changed solubility of cholesterol in the micelle system. SM has been shown to facilitate the solubility of cholesterol [37] whereas formation of ceramide in bilayers may drive cholesterol from bilayer order phase to a crystal phase, thus decrease the solubility of cholesterol [38]. Further biophysical studies are required to disclose the mechanism.…”