The effects of cholesterol and β-sitosterol on the structure of saturated diacylphosphatidylcholine bilayers

The structures of DMPC and DPPC bilayers in unilamellar liposomes, in the presence of 33.3 mol% cholesterol or the plant sterol β-sitosterol, have been studied by small-angle neutron scattering. The bilayer thickness d _L increases in a similar way for both sterols. The repeat distance in multilamellar liposomes, as determined by small-angle X-ray diffraction, is larger in the presence of β-sitosterol than in the presence of cholesterol. We observe that each sterol modifies the interlamellar water layer differently, cholesterol reducing its thickness more efficiently than β-sitosterol, and conclude that cholesterol suppresses bilayer undulations more effectively than β-sitosterol.     

A comparative calorimetric and spectroscopic study of the effects of cholesterol and of the plant sterols β-sitosterol and stigmasterol on the thermotropic phase behavior and organization of dipalmitoylphosphatidylcholine bilayer membranes

We performed comparative DSC and FTIR spectroscopic measurements of the effects of β-sitosterol (Sito) and stigmasterol (Stig) on the thermotropic phase behavior and organization of DPPC bilayers. Sito and Stig are the major sterols in the biological membranes of higher plants, whereas cholesterol (Chol) is the major sterol in mammalian membranes. Sito differs in structure from Chol in having an ethyl group at C24 of the alkyl side-chain, and Stig in having both the C24 ethyl group and trans-double bond at C22. Our DSC studies indicate that the progressive incorporation of Sito and Stig decrease the temperature and cooperativity of the pretransition of DPPC to a slightly lesser and greater extent than Chol, respectively, but the pretransition persists to 10 mol % sterol concentration in all cases. All three sterols produce essentially identical effects on the thermodynamic parameters of the sharp component of the DPPC main phase transition. However, the ability to increase the temperature and decrease the cooperativity and enthalpy of the broad component decreases in the order Chol > Sito > Stig. Nevertheless, at higher Sito/Stig concentrations, there is no evidence of sterol crystallites. Our FTIR spectroscopic studies demonstrate that Sito and especially Stig incorporation produces a smaller ordering of the hydrocarbon chains of fluid DPPC bilayers than does Chol. In general, the presence of a C24 ethyl group in the alkyl side-chain reduces the characteristic effects of Chol on the thermotropic phase behavior and organization of DPPC bilayer membranes, and a trans-double bond at C22 magnifies this effect.     

Localization of VE-cadherin in plasmalemmal cholesterol rich microdomains and the effects of cholesterol depletion on VE-cadherin mediated cell–cell adhesion

VE-cadherin is the predominant adhesion molecule in vascular endothelial cells being responsible for maintenance of the endothelial barrier function by forming adhesive contacts (adherens junctions) to neighbouring cells. We found by use of single molecule fluorescence microscopy that VE-cadherin is localised in preformed clusters when not inside adherens junctions. These clusters depend on the integrity of the actin cytoskeleton and are localised in cholesterol rich microdomains of mature endothelial cells as found by membrane fractionation. The ability to form and maintain VE-cadherin based junctions was probed using the laser tweezer technique, and we found that cholesterol depletion has dramatical effects on VE-cadherin mediated adhesion. While a 30% reduction of the cholesterol-level results in an increase of adhesion, excessive cholesterol depletion by about 60% leads to an almost complete loss of VE-cadherin function. Nevertheless, the cadherin concentration in the membrane and the single molecule kinetic parameters of the cadherin are not changed. Our results suggest that the actin cytoskeleton, junction-associated proteins and protein–lipid assemblies in cholesterol-rich microdomains mutually stabilise each other to form functional adhesion contacts.     

The effect of cholesterol and gramicidin A′ on the carbonyl groups of dimyristoylphosphatidylcholine dispersions

Proton-enhanced carbon-13 magnetic resonance measurements have been made of the natural abundance carbon-13 carbons in hydrated L_α phase dispersions of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) codispersed with cholesterol or with the polypeptide gramicidin A′. The carbonyl group spectrum consists of a superposition of two peaks derived from the two carbonyl sites within the lipid. In the L_α phase of DMPC both carbonyl sites contribute axially symmetric spectra, one with a chemical shift anisotropy of –29 ppm and the other with a chemical shift anisotropy of less than –5 ppm. The chemical shift anisotropy of the broader carbonyl resonance was found to increase with increasing cholesterol content. However, in DMPC dispersions with gramicidin A′, the chemical shift anisotropy of the broader carbonyl signal initially increased slightly from that of pure DMPC and then decreased with increasing concentrations of gramicidin A′. The width of the narrower spectral component was essentially unaltered by cholesterol or gramicidin A′. The presence of a narrow component at all concentrations of cholesterol or gramicidin A′ suggests that it is unlikely that any significant conformational changes have occurred at the carbonyl level of the bilayer. We propose that the major effect of cholesterol or gramicidin A′ is to alter the molecular order parameter, S_mol, which reflects the range of angles through which the local molecular long axis of the phospholipid is tumbling.     

Modulation of 3-hydroxy-3-methylglutaryl-CoA reductase and of acyl-CoA–cholesterol acyltransferase by the transfer of non-esterified cholesterol to rat liver microsomal vesicles

The incubation of rat liver microsomal fraction with a serum preparation followed by the re-isolation of the microsomal membranes has resulted in an increase in the concentration of non-esterified cholesterol, a considerable decrease in the activity of 3-hydroxy-3-methylglutaryl-CoA reductase and in an increase in the activity of acyl-CoA–cholesterol acyltransferase in the treated microsomal preparation. These effects were related to the concentration of serum in the incubation mixture and to the duration of the incubation. The transfer of non-esterified cholesterol was specific in that the content of protein and the total phospholipids were similar in the original microsomal fraction and the serum-treated microsomal preparation. The incubation of the microsomal fraction with lipoprotein-deficient serum or with no serum resulted in both cases in small changes in the non-esterified cholesterol, the esterified cholesterol and the total phospholipid content in the treated preparations compared with these concentrations in the original microsomal fraction, whereas the activity of acyl-CoA–cholesterol acyltransferase and of 3-hydroxy-3-methylglutaryl-CoA reductase was similar in the lipoprotein-deficient-serum-treated and the buffer-treated microsomal preparations. The activity of 3-hydroxy-3-methylglutaryl-CoA reductase was lower and the activity of acyl-CoA–cholesterol acyltransferase was higher in the lipoprotein-deficient-serum-treated and the buffer-treated microsomal preparations as compared with these activities in the original microsomal fraction. However, the serum-treated microsomal preparation had considerably lower activity of 3-hydroxy-3-methylglutaryl-CoA reductase and considerably higher activity of acyl-CoA–cholesterol acyltransferase than these activities in buffer-treated and in lipoprotein-deficient-serum-treated microsomal preparations.     

HDL and cholesterol: life after the divorce?

For decades, HDL and HDL-cholesterol (HDL-C) levels were viewed as synonymous, and modulation of HDL-C levels by drug therapy held great promise for the prevention and treatment of cardiovascular disease. Nevertheless, recent failures of drugs that raise HDL-C to reduce cardiovascular risk and the now greater understanding of the complexity of HDL composition and biology have prompted researchers in the field to redefine HDL. As such, the focus of HDL has now started to shift away from a cholesterol-centric view toward HDL particle number, subclasses, and other alternative metrics of HDL. Many of the recently discovered functions of HDL are, in fact, not strictly conferred by its ability to promote cholesterol flux but by the other molecules it transports, including a diverse set of proteins, small RNAs, hormones, carotenoids, vitamins, and bioactive lipids. Based on HDL''s ability to interact with almost all cells and transport and deliver fat-soluble cargo, HDL has the remarkable capacity to affect a wide variety of endocrine-like systems. In this review, we characterize HDL''s unique cargo and address the functional relevance and consequences of HDL transport and delivery of noncholesterol molecules to recipient cells and tissues.     

Effect of cholesterol and dipalmitoyl phosphatidylcholine enrichment on the kinetics of Na−Li exchange of human erythrocytes

Summary The effects of cholesterol loading and depletion and of a 10% replacement of native phosphatidylcholine by dipalmitoyl phosphatidylcholine (di 16:0-PC) on kinetic properties of human red cell Na–Li exchange have been studied.Compared to control erythrocytes (cholesterol/phospholipid ratio (C/P=0.8–0.9)),V _max of phloretin-sensitive Li uptake and of Li efflux stimulated by extracellular Na (Na _o ) were reduced by 15–30% in cholesterol-loaded red cells (C/P=1.05–1.33). The apparentK _m values for external Li (Li _o ) and for internal Li (Li _i ) were decreased by about one-third in these cells. Cholesterol depletion (C/P=0.7) exerted opposite effects on the kinetics of Na _o -dependent Li efflux. On augmentingC/P from 0.66 to 1.0,V _max of Na _o -dependent Li efflux was reduced by about 30%; increasingC/P above 1.0 caused no further lowering ofV _max. Li leakage rates monotonically decreased over the whole range ofC/P ratios examined (0.66–1.3). This indicates that Na–Li exchange and Li leak are differentially affected by cholesterol.Incorporation of di 16:0-PC (replacement of 3% of total red cell phospholipids) caused similar kinetic alterations of Na–Li exchange as a rise in membrane cholesterol by 20–50%. Notably, selective incorporation of di 16:0-PC into the outer monolayer increased both intra- and extracellular Li binding affinities of Na–Li exchange and lowered its maximum velocity. Thus, both di 16:0-PC enrichment and cholesterol loading exerted an uncompetitive type of transport inhibition. The results are in agreement with the hypothesis that the kinetic alterations of red cell Na–Li exchange seen in a subgroup of essential hypertensive patients could be due to subtle changes in the molecular species composition of individual phospholipids.     

Niemann–Pick C1-Like 1 and cholesterol uptake

Niemann–Pick C1-Like 1 (NPC1L1) is a polytopic transmembrane protein responsible for dietary cholesterol and biliary cholesterol absorption. Consistent with its functions, NPC1L1 distributes on the brush border membrane of enterocytes and the canalicular membrane of hepatocytes in humans. As the molecular target of ezetimibe, a hypocholesterolemic drug, its physiological and pathological significance has been recognized and intensively studied for years. Recently, plenty of new findings reveal the molecular mechanism of NPC1L1's role in cholesterol uptake, which may provide new insights on our understanding of cholesterol absorption. In this review, we summarized recent progress in these studies and proposed a working model, hoping to provide new perspectives on the regulation of cholesterol transport and metabolism.     

Direct evidence of abca1-mediated efflux of cholesterol at the mouse blood�Cbrain barrier

We investigated the expression and function of Abca1 in wild-type C57BL/6, abca1(+/+), and abca1(-/-) mice brain capillaries forming the blood-brain barrier (BBB). We first demonstrated by quantitative RT-PCR and Western immunoblot that Abca1 was expressed and enriched in the wild-type mouse brain capillaries. In abca1(-/-) mice, we reported that the lack of Abca1 resulted in an 1.6-fold increase of the Abcg4 expression level compared to abca1(+/+) mice. Next, using the in situ brain perfusion technique, we showed that the [(3)H]cholesterol brain uptake clearance (Cl(up), μl/s/g brain), was significantly increased (107%) in abca1(-/-) mice compared to abca1(+/+) mice, meaning that the deficiency of Abca1 conducted to a significant decrease of the cholesterol efflux at the BBB level. In addition, the co-perfusion of probucol (Abca1 inhibitor) with [(3)H]cholesterol resulted in an increase of [(3)H]cholesterol Cl(up) (115%) in abca1(+/+) but not in abca1(-/-) mice, meaning that probucol inhibited selectively the efflux function of Abca1. In conclusion, our results demonstrated that Abca1 was expressed in the mouse brain capillaries and that Abca1 functions as an efflux transporter through the mouse BBB.     

Effects of a cholesterol esterase inhibitor and of prostaglandin F2α on testis cholesterol and on plasma testosterone in mice

Administration of Phenylmethylsulfonylfluoride, an inhibitor of cholesterol esterase, to male mice caused an increase in the concentration of esterified cholesterol in the testis, a decrease in the weight of seminal vesicles and a decrease in the concentration of testosterone in peripheral plasma. It is suggested that hydrolysis of cholesterol esters present in the testis is required for normal production of androgenic steroids.Administration of prostaglandin F_2α to male mice lowered plasma testosterone levels and raised the concentration of esterified cholesterol in the testes. Apparently testicular steroidogenesis was inhibited.     

Methyl-β-cyclodextrin restores the structure and function of pulmonary surfactant films impaired by cholesterol

Pulmonary surfactant, a defined mixture of lipids and proteins, imparts very low surface tension to the lung-air interface by forming an incompressible film. In acute respiratory distress syndrome and other respiratory conditions, this function is impaired by a number of factors, among which is an increase of cholesterol in surfactant. The current study shows in vitro that cholesterol can be extracted from surfactant and function subsequently restored to dysfunctional surfactant films in a dose-dependent manner by methyl-β-cyclodextrin (MβCD). Bovine lipid extract surfactant was supplemented with cholesterol to serve as a model of dysfunctional surfactant. Likewise, when cholesterol in a complex with MβCD (“water-soluble cholesterol”) was added in aqueous solution, surfactant films were rendered dysfunctional. Atomic force microscopy showed recovery of function by MβCD is accompanied by the re-establishment of the native film structure of a lipid monolayer with scattered areas of lipid bilayer stacks, whereas dysfunctional films lacked bilayers. The current study expands upon a recent perspective of surfactant inactivation in disease and suggests a potential treatment.     

Spectroscopic and calorimetric studies on trazodone hydrochloride–phosphatidylcholine liposome interactions in the presence and absence of cholesterol

The interaction of antidepressant drug trazodone hydrochloride (TRZ) with dipalmitoyl phosphatidylcholine (DPPC) multilamellar liposomes (MLVs) in the presence and absence of cholesterol (CHO) was investigated as a function of temperature by using Electron Paramagnetic Resonance (EPR) spin labeling, Fourier Transform Infrared (FTIR) Spectroscopy and Differential Scanning Calorimetry (DSC) techniques. These interactions were also examined for dimyristoyl phosphatidylcholine (DMPC) multilamellar liposomes by using Electron Paramagnetic Resonance (EPR) spin labeling technique. In the EPR spin labeling studies, 5- and 16-doxyl stearic acid (5-DS and 16-DS) spin labels were used to monitor the head group and alkyl chain region of phospholipids respectively. The results indicated that TRZ incorporation causes changes in the physical properties of PC liposomes by decreasing the main phase transition temperature, abolishing the pre-transition, broadening the phase transition profile, and disordering the system around the head group region. The interaction of TRZ with unilamellar (LUV) DPPC liposomes was also examined. The most pronounced effect of TRZ on DPPC LUVs was observed as the further decrease of main phase transition temperature in comparison with DPPC MLVs. The mentioned changes in lipid structure and dynamics caused by TRZ may modulate the biophysical activity of membrane associated receptors and in turn the pharmacological action of TRZ.     

Similar effects ofβ-alanine and taurine in cholesterol metabolism

β-Alanine, though producing a deficiency of taurine in the tissues, had a similar effect on cholesterol metabolism as taurine. Both caused increased activity of hepatic hydroxymethylglutaryl coenzyme A reductase and increased incorporation of 1, 2 of [¹⁴C]-acetate into liver cholesterol. Both caused increased concentration of biliary cholesterol and bile acids. There was increased activity of lipoprotein lipase in heart, but decreased activity in the adipose tissue in both cases. Release of lipoproteins into circulation was decreased in both cases.     

Interleukin-1�� enhances the intracellular accumulation of cholesterol by up-regulating the expression of low-density lipoprotein receptor and 3-hydroxy-3-methylglutaryl coenzyme A reductase in podocytes

The aim of this article is to investigate whether interleukin-1β (IL-1β) could regulate the intracellular accumulation of cholesterol and the expression of lipid-metabolism-related regulators in podocytes in vitro and the potential mechanisms. Podocytes were treated with 200?μg/ml of low-density protein (LDL), 20?ng/ml of IL-1β, or 200?μg/ml of LDL plus 5-20?ng/ml of IL-1β for 24?h in vitro. The contents of intracellular cholesterol were determined by enzymatic assays and Oil Red O staining. The levels of LDL receptor (LDLr), 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, sterol regulatory element binding protein 2 (SREBP-2), SREBP cleavage activating protein (SCAP), and insulin-induced gene-1 (Insig-1) expression were characterized by real-time polymerase chain reaction (RT-PCR) and Western blot assays. Treatment with IL-1β or LDL alone increased the contents of intracellular cholesterol (P?相似文献   

Crystal Structures of the Network-Forming Short-Arm Tips of the Laminin β1 and γ1 Chains

The heterotrimeric laminins are a defining component of basement membranes and essential for tissue formation and function in all animals. The three short arms of the cross-shaped laminin molecule are composed of one chain each and their tips mediate the formation of a polymeric network. The structural basis for laminin polymerisation is unknown. We have determined crystal structures of the short-arm tips of the mouse laminin β1 and γ1 chains, which are grossly similar to the previously determined structure of the corresponding α5 chain region. The short-arm tips consist of a laminin N-terminal (LN) domain that is attached like the head of a flower to a rod-like stem formed by tandem laminin-type epidermal growth factor-like (LE) domains. The LN domain is a β-sandwich with elaborate loop regions that differ between chains. The γ1 LN domain uniquely contains a calcium binding site. The LE domains have little regular structure and are stabilised by cysteines that are disulphide-linked 1-3, 2-4, 5-6 and 7-8 in all chains. The LN surface is not conserved across the α, β and γ chains, but within each chain subfamily there is a striking concentration of conserved residues on one face of the β-sandwich, while the opposite face invariably is shielded by glycans. We propose that the extensive conserved patches on the β and γ LN domains mediate the binding of these two chains to each other, and that the α chain LN domain subsequently binds to the composite β-γ surface. Mutations in the laminin β2 LN domain causing Pierson syndrome are likely to impair the folding of the β2 chain or its ability to form network interactions.     

Changes in the Anatomic and Microscopic Structure and the Expression of HIF-1α and VEGF of the Yak Heart with Aging and Hypoxia

The study aimed to identify the changes of anatomic and microscopic structure and the expression and localization of hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) in the myocardium and coronary artery of the yak heart adapted to chronic hypoxia with aging. Thirty-two yaks (1 day, 6 months, 1 year, 2 years, and 5 year old) were included, and immunoelectronmicroscopy, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA) were used. Right ventricular hypertrophy was not present in yaks with aging. There was no intima thickening phenomenon in the coronary artery. The ultrastructure of myofibrils, mitochondria, and collagen fibers and the diameter and quantity of collagen changed significantly with aging. The enzymatic activity of complexes I, II, and V increased with age. Immunogold labeling showed the localization of HIF-1α protein in the cytoplasm and nuclei of endothelial cells and cytoplasm of cardiac muscle cells, and VEGF protein in the nuclei and perinuclei areas of smooth muscle cells of coronary artery, and in the cytoplasm and nuclei of endothelial cells. ELISA results showed that HIF-1α secretion significantly increased in the myocardium and coronary artery from an age of 1 day to 2 years of yaks and decreased in old yaks. However, VEGF protein always increased with aging. The findings of this study suggest that 6 months is a key age of yak before which there are some adaptive changes to deal with low-oxygen environment, and there is a maturation of the yak heart from the age of 6 months to 2 years.     

22(R)-hydroxycholesterol and pioglitazone synergistically decrease cholesterol ester via the PPARγ–LXRα–ABCA1 pathway in cholesterosis of the gallbladder

Cholesterosis is a disease of cholesterol metabolism characterized by the presence of excessive lipid droplets in the cytoplasm. These lipid droplets are mainly composed of cholesterol esters derived from free cholesterol. The removal of excess cholesterol from gallbladder epithelial cells (GBECs) is very important for the maintenance of intracellular cholesterol homeostasis and the preservation of gallbladder function. Several lines of evidence have indicated that the activation of either peroxisome proliferator-activated receptor gamma (PPARγ) or liver X receptor α (LXRα) relates to cholesterol efflux. While pioglitazone can regulate the activation of PPARγ, 22(R)-hydroxycholesterol can activate LXRα and is a metabolic intermediate in the biosynthesis of steroid hormones. However, the effect of 22(R)-hydroxycholesterol in combination with pioglitazone on cholesterosis of the gallbladder is unclear. GBECs were treated with pioglitazone, 22(R)-hydroxycholesterol or PPARγ siRNA followed by Western blot analysis for ATP-binding cassette transporter A1 (ABCA1), PPARγ and LXRα. Cholesterol efflux to apoA-I was determined, and Oil Red O staining was performed to monitor variations in lipid levels in treated GBECs. Our data showed that 22(R)-hydroxycholesterol can modestly up-regulate LXRα while simultaneously increasing ABCA1 by 56%. The combination of 22(R)-hydroxycholesterol and pioglitazone resulted in a 3.64-fold increase in ABCA1 expression and a high rate of cholesterol efflux. Oil Red O staining showed an obvious reduction in the lipid droplets associated with cholesterosis in GBECs. In conclusion, the present findings indicate that the anti-lipid deposition action of 22(R)-hydroxycholesterol combined with pioglitazone involves the activation of the PPARγ–LXRα–ABCA1 pathway, increased ABCA1 expression and the efflux of cholesterol from GBECs. Thus, 22(R)-hydroxycholesterol synergistically combined with pioglitazone to produce a remarkable effect on lipid deposition in cholesterosis GBECs.     

Clathrin-dependent APP endocytosis and Aβ secretion are highly sensitive to the level of plasma membrane cholesterol

Several lines of evidence support a strong relationship between cholesterol and Alzheimer's disease pathogenesis. Membrane cholesterol is known to modulate amyloid precursor protein (APP) endocytosis and amyloid-β (Aβ) secretion. Here we show in a human cell line model of endocytosis (HEK293 cells) that cholesterol exerts these effects in a dose-dependent and linear manner, over a wide range of concentrations (-40% to + 40% variations of plasma membrane cholesterol induced by methyl-beta-cyclodextrin (MBCD) and MBCD-cholesterol complex respectively). We found that the gradual effect of cholesterol is inhibited by small interference RNA-mediated downregulation of clathrin. Modulation of clathrin-mediated APP endocytosis by cholesterol was further demonstrated using mutants of proteins involved in the formation of early endosomes (dynamin2, Eps15 and Rab5). Importantly we show that membrane proteins other than APP are not affected by cholesterol to the same extent. Indeed clathrin-dependent endocytosis of transferrin and cannabinoid1 receptors as well as internalization of surface proteins labelled with a biotin derivative (sulfo-NHS-SS-biotin) were not sensitive to variations of plasma membrane cholesterol from -40% to 40%. In conclusion clathrin-dependent APP endocytosis appears to be very sensitive to the levels of membrane cholesterol. These results suggest that cholesterol increase in AD could be responsible for the enhanced internalization of clathrin-, dynamin2-, Eps15- and Rab5-dependent endocytosis of APP and the ensuing overproduction of Aβ.