Free fatty acids modulate intermembrane trafficking of cholesterol by increasing lipid mobilities:: Novel 13C NMR analyses of free cholesterol partitioning

Cholesterol and free fatty acids in membranes modulate major biological processes, and their cellular metabolism and actions are often coordinately regulated. However, effects of free fatty acid on cholesterol-membrane interactions have proven difficult to monitor in real time in intact systems. We developed a novel C-13 NMR method to assess effects of free fatty acids on molecular interactions of cholesterol within-and transfer between-model membranes. An important advantage of this method is the ability to acquire kinetic data without separation of donor and acceptor membranes. Large unilamellar phospholipid vesicles (LUV) with phosphatidylcholine/cholesterol ratios of 4:1 served as cholesterol donors. Small unilamellar vesicles (SUV) made with phosphatidylcholine were acceptors. The C-13(4)-cholesterol peak is narrow in SUV, but very broad in LUV, spectra; the increase in intensity of this peak over time monitored transfer. Oleic acid and other long chain free fatty acids [saturated (C12-18) and unsaturated (C18)] dose-dependently increased mobilities of lipids in LUV (phospholipid and cholesterol) and cholesterol transfer rates, whereas short (C8-10) and very long (C24) chain free fatty acids did not. Decreasing pH from 7.4 to 6.5 (+/-oleic acid) had no effect on cholesterol transfer, and 5 mol % fatty acyl-CoAs increased transfer rates, demonstrating greater importance of the fatty-acyl tail over the headgroup. In LUV containing sphingomyelin, transfer rates decreased, but the presence of oleic acid increased transfer 1.3-fold. These results demonstrate free fatty acid-facilitated cholesterol movement within and between membranes, which may contribute to their multiple biological effects.