Fatty acids are rapidly delivered to and extracted from membranes by methyl-β-cyclodextrin

摘要

We performed detailed biophysical studies of transfer of long-chain fatty acids (FAs) from methyl-β-CD (MBCD) to model membranes (egg-PC vesicles) and cells and the extraction of FA from membranes by MBCD. We used i) fluorescein phosphatidylethanolamine to detect transfer of FA anions arriving in the outer membrane leaflet; ii) entrapped pH dyes to measure pH changes after FA diffusion (flip-flop) across the lipid bilayer; and iii) soluble fluorescent-labeled FA binding protein to measure the concentration of unbound FA in water. FA dissociated from MBCD, bound to the membrane, and underwent flip-flop within milliseconds. In the presence of vesicles, MBCD maintained the aqueous concentration of unbound FA at low levels comparable to those measured with albumin. In studies with cells, addition of oleic acid (OA) complexed with MBCD yielded rapid (seconds) dose-dependent OA transport into 3T3-L1 preadipocytes and HepG2 cells. MBCD extracted OA from cells and model membranes rapidly at concentrations exceeding those required for OA delivery but much lower than concentrations commonly used for extracting cholesterol. Compared with albumin, MBCD can transfer its entire FA load and is less likely to extract cell nutrients and to introduce impurities.