Multidrug resistance P-glycoprotein is not involved in cholesterol esterification

The aim of the present paper is to reinvestigate the role of multidrug resistance P-glycoprotein MDR1 and MDR-associated protein (MRP1) in cholesterol esterification using well-characterized inhibitors. Using specific substrate efflux assay, we show that GF120918 (0.2 microM) and probenecid (5 mM) were specific inhibitors of MDR1 and MRP1, respectively. In HepG2 cells, neither of them affect the esterification of cholesterol derived from the uptake of cholesterol-rich lipoprotein, while both verapamil (100 microM) and progesterone (100 microM) were able to inhibit cholesterol esterification. Similar results were obtained with verapamil, progesterone, and GF120918 in the MDR1-overexpressing cells MCF7/ADR. The capacity of progesterone to reduce cholesterol esterification is not correlated with its ability to inhibit MDR1 but is rather due to direct inhibition of acyl-CoA:cholesterol acyltransferase (ACAT). We conclude that the esterification of cholesterol is not correlated with MDR1 or MRP1 activity, thus excluding their role in the intracellular transport of endocytosis-derived cholesterol.     

Phosphatidylinositol promotes cholesterol transport and excretion

Administration of phosphatidylinositol (PI) to New Zealand White rabbits increases HDL negative charge and stimulates reverse cholesterol transport. Intravenously administered PI (10 mg/kg) associated almost exclusively with the HDL fraction in rabbits. PI promoted an increase in the hepatic uptake of plasma free cholesterol (FC) and a 21-fold increase in the biliary secretion of plasma-derived cholesterol. PI also increased cholesterol excretion into the feces by 2.5-fold. PI directly affects cellular cholesterol metabolism. In cholesterol-loaded macrophages, PI stimulated cholesterol mass efflux to lipid-poor reconstituted HDL. PI was about half as effective as cAMP at stimulating efflux, and the effects of cAMP and PI were additive. In cultured HepG2 cells, PI-enriched HDL also enhanced FC uptake from HDL by 3-fold and decreased cellular cholesterol synthesis and esterification. PI enrichment had no effect on the selective uptake of cholesterol esters or on the internalization of HDL particles. PI-dependent metabolic events were efficiently blocked by inhibitors of protein kinase C and the inositol signaling cascade.The data suggest that HDL-PI acts via cell surface ATP binding cassette transporters and signaling pathways to regulate both cellular and intravascular cholesterol homeostasis.     

Sterol-binding proteins and endosomal cholesterol transport

Endosomal compartments sort and deliver exogenous lipoprotein-derived cholesterol to the endoplasmic reticulum for regulating cellular cholesterol homeostasis. A large number of studies have focused on the removal of endosomal cholesterol, since its accumulation leads to devastating human diseases. Recent studies suggest that cytoplasmic sterol-binding proteins may be involved in endosomal cholesterol transport. In particular, endosome/lysosome-localized or -associated cholesterol-binding proteins may serve as key mediators of cholesterol removal in a non-vesicular manner. Further characterization of these cholesterol-binding proteins will shed light on the molecular mechanisms that regulate endosomal cholesterol sorting.     

Is reverse cholesterol transport regulated by active cholesterol?

This review considers the hypothesis that a small portion of plasma membrane cholesterol regulates reverse cholesterol transport in coordination with overall cellular homeostasis. It appears that almost all of the plasma membrane cholesterol is held in stoichiometric complexes with bilayer phospholipids. The minor fraction of cholesterol that exceeds the complexation capacity of the phospholipids is called active cholesterol. It has an elevated chemical activity and circulates among the organelles. It also moves down its chemical activity gradient to plasma HDL, facilitated by the activity of ABCA1, ABCG1, and SR-BI. ABCA1 initiates this process by perturbing the organization of the plasma membrane bilayer, thereby priming its phospholipids for translocation to apoA-I to form nascent HDL. The active excess sterol and that activated by ABCA1 itself follow the phospholipids to the nascent HDL. ABCG1 similarly rearranges the bilayer and sends additional active cholesterol to nascent HDL, while SR-BI simply facilitates the equilibration of the active sterol between plasma membranes and plasma proteins. Active cholesterol also flows downhill to cytoplasmic membranes where it serves both as a feedback signal to homeostatic ER proteins and as the substrate for the synthesis of mitochondrial 27-hydroxycholesterol (27HC). 27HC binds the LXR and promotes the expression of the aforementioned transport proteins. 27HC-LXR also activates ABCA1 by competitively displacing its inhibitor, unliganded LXR. 4 Considerable indirect evidence suggests that active cholesterol serves as both a substrate and a feedback signal for reverse cholesterol transport. Direct tests of this novel hypothesis are proposed.     

The role of intracellular cholesterol transport in cholesterol homeostasis

How cholesterol is transported among the membranes of the cell is obscure. Similarly, the mechanisms governing the abundance of cell cholesterol are not entirely understood. It may be, however, that a link exists between the intracellular transport of cholesterol and its homeostasis. We propose that cholesterol circulates between the plasma membrane, which contains the bulk of the sterol, and organelle membranes, which contain only traces. A putative sensor translates small fluctuations in plasma membrane cholesterol into relatively large changes in this flux, thereby setting the magnitude of the intracellular pools. The cholesterol concentration in the endoplasmic reticulum and mitochondrial membranes then governs the activities of proteins embedded therein that mediate cholesterol transformations. This arrangement creates a feedback loop through which the intracellular effectors regulate the abundance of plasma membrane cholesterol.     

NPC1L1 and cholesterol transport

The polytopic transmembrane protein, Niemann-Pick C1-Like 1 (NPC1L1), is enriched in the apical membrane of small intestine absorptive enterocytes where it mediates extracellular sterol transport across the brush border membrane. It is essential for intestinal sterol absorption and is the molecular target of ezetimibe, a potent cholesterol absorption inhibitor that lowers blood cholesterol in humans. NPC1L1 is also highly expressed in human liver. The hepatic function of NPC1L1 may be to limit excessive biliary cholesterol loss. NPC1L1-dependent sterol uptake seems to be a clathrin-mediated endocytic process and is regulated by cellular cholesterol content. Recently, NPC1L1 inhibition has been shown to have beneficial effects on components of the metabolic syndrome, such as obesity, insulin resistance, and fatty liver, in addition to atherosclerosis.     

Effect of triarimol on cholesterol biosynthesis in rat-liver subcellular fractions

Cholesterol biosynthesis was studied in rat-liver subcellular fractions incubated with DL-[2-¹⁴C]mevalonic acid in the presence and absence of triarimol (α-(2,4-dichlorophenyl)-α-phenyl-5-pyrimidine methanol). Triarimol strongly inhibits incorporation of radioactivity into cholesterol and this results in a large accumulation of radioactive lanosterol and 24,25-dihydro-lanosterol. The inhibition of lanosterol 14α-demethylase by triarimol was confirmed by assay of the enzyme in rat-liver microsomal fraction in the presence and absence of the inhibitor. Apart from a slight inhibition of Δ⁷-sterol-Δ⁵-dehydrogenase, triarimol did not affect the activity of any other enzyme involved in cholesterol biosynthesis from acetate.     

胆固醇逆向转运的分子机制

胆固醇逆向转运是周围细胞胆固醇转运至肝脏转化、清除的重要生理过程,它在维持机体胆固醇代谢平衡和对抗动脉粥样硬化发生及发展中起重要作用。研究证实胆固醇逆向转运直是高密度脂蛋白在多种生物活性分子参与下,由新生前β－ＨＤＬ到成熟α－ＨＤＬ递变的胆固醇转运及代谢过程。