Sensitivity of Volume-regulated Anion Current to Cholesterol Structural Analogues

Depletion of membrane cholesterol and substitution of endogenous cholesterol with its structural analogues was used to analyze the mechanism by which cholesterol regulates volume-regulated anion current (VRAC) in endothelial cells. Depletion of membrane cholesterol enhanced the development of VRAC activated in a swelling-independent way by dialyzing the cells either with GTPγS or with low ionic strength solution. Using MβCD–sterol complexes, 50–80% of endogenous cholesterol was substituted with a specific analogue, as verified by gas-liquid chromatography. The effects of cholesterol depletion were reversed by the substitution of endogenous cholesterol with its chiral analogue, epicholesterol, or with a plant sterol, β-sitosterol, two analogues that mimic the effect of cholesterol on the physical properties of the membrane bilayer. Alternatively, when cholesterol was substituted with coprostanol that has only minimal effect on the membrane physical properties it resulted in VRAC enhancement, similar to cholesterol depletion. In summary, our data show that these channels do not discriminate between the two chiral analogues of cholesterol, as well as between the two cholesterols and β-sitosterol, but discriminate between cholesterol and coprostanol. These observations suggest that endothelial VRAC is regulated by the physical properties of the membrane

Modulation of Endothelial Inward-Rectifier K+ Current by Optical Isomers of Cholesterol

Membrane potential of aortic endothelial cells under resting conditions is dominated by inward-rectifier K+ channels belonging to the Kir 2 family. Regulation of endothelial Kir by membrane cholesterol was studied in bovine aortic endothelial cells by altering the sterol composition of the cell membrane. Our results show that enriching the cells with cholesterol decreases the Kir current density, whereas depleting the cells of cholesterol increases the density of the current. The dependence of the Kir current density on the level of cellular cholesterol fits a sigmoid curve with the highest sensitivity of the Kir current at normal physiological levels of cholesterol. To investigate the mechanism of Kir regulation by cholesterol, endogenous cholesterol was substituted by its optical isomer, epicholesterol. Substitution of ~50% of cholesterol by epicholesterol results in an early and significant increase in the Kir current density. Furthermore, substitution of cholesterol by epicholesterol has a stronger facilitative effect on the current than cholesterol depletion. Neither single channel properties nor membrane capacitance were significantly affected by the changes in the membrane sterol composition. These results suggest that 1) cholesterol modulates cellular K+ conductance by changing the number of the active channels and 2) that specific cholesterol-protein interactions are critical for the regulation of endothelial Kir

Hepatic Proprotein Convertases Modulate HDL Metabolism

SummaryThe risk of atherosclerosis is inversely associated with plasma levels of high-density lipoprotein cholesterol (HDL-C). However, HDL metabolism is incompletely understood, and there are few effective approaches to modulate HDL-C levels. Here we show that inhibition in the liver of the classical proprotein convertases (PCs), but not the atypical PCs S1P and PCSK9, decreases plasma HDL-C levels. This metabolic effect of hepatic PCs is critically dependent on expression of endothelial lipase (EL), an enzyme that directly hydrolyzes HDL phospholipids and promotes its catabolism. Hepatic PCs reduce EL function through direct inactivating cleavage of EL as well as through activating cleavage of angiopoietin-like protein 3 (ANGPTL3), an endogenous inhibitor of EL. Thus, inhibition of hepatic PCs results in increased EL activity, leading to reduced HDL-C as well as impaired reverse cholesterol transport. The hepatic PC–ANGPTL3–EL–HDL pathway is therefore a novel mechanism controlling HDL metabolism and cholesterol homeostasis

The Effects of Apolipoprotein F Deficiency on High Density Lipoprotein Cholesterol Metabolism in Mice

Apolipoprotein F (apoF) is 29 kilodalton secreted sialoglycoprotein that resides on the HDL and LDL fractions of human plasma. Human ApoF is also known as Lipid Transfer Inhibitor protein (LTIP) based on its ability to inhibit cholesteryl ester transfer protein (CETP)-mediated transfer events between lipoproteins. In contrast to other apolipoproteins, ApoF is predicted to lack strong amphipathic alpha helices and its true physiological function remains unknown. We previously showed that overexpression of Apolipoprotein F in mice reduced HDL cholesterol levels by 20–25% by accelerating clearance from the circulation. In order to investigate the effect of physiological levels of ApoF expression on HDL cholesterol metabolism, we generated ApoF deficient mice. Unexpectedly, deletion of ApoF had no substantial impact on plasma lipid concentrations, HDL size, lipid or protein composition. Sex-specific differences were observed in hepatic cholesterol content as well as serum cholesterol efflux capacity. Female ApoF KO mice had increased liver cholesteryl ester content relative to wild type controls on a chow diet (KO: 3.4+/−0.9 mg/dl vs. WT: 1.2+/−0.3 mg/dl, p<0.05). No differences were observed in ABCG1-mediated cholesterol efflux capacity in either sex. Interestingly, ApoB-depleted serum from male KO mice was less effective at promoting ABCA1-mediated cholesterol efflux from J774 macrophages relative to WT controls