The selective uptake of high density lipoprotein cholesterol esters by
the liver : the role of scavenger receptor BI in reverse cholesterol
transport
Fluiter, K.
Citation
Fluiter, K. (1998, June 23). The selective uptake of high density lipoprotein cholesterol
esters by the liver : the role of scavenger receptor BI in reverse cholesterol transport.
Retrieved from https://hdl.handle.net/1887/5437
Version:
Corrected Publisher’s Version
License:
Licence agreement concerning inclusion of doctoral thesis in the
Institutional Repository of the University of Leiden
liver
The selective uptake of high density lipoprotein cholesterol esters by the
liver
The role of scavenger receptor in reverse cholesterol transport
proefschrift
ter verkrijging van de graad van Doctor aan de Rijksuniversiteit te Leiden,
op gezag van de Rector Magnificus Dr. W.A. Wagenaar, hoogleraar in de faculteit der Sociale Wetenschappen,
volgens besluit van het College voor Promoties, te verdedigen op dinsdag 23 juni 1998
te klokke uur
door
Kees Fluiter
Referent: Prof. dr. ir. L. M. Havekes
Overige leden: Prof. dr. van der (Universiteit van Kentucky, U.S.A.) Prof. dr. D. Roos (Universteit van Amsterdam)
Prof. dr. D.D. Breimer Dr. ir. E.A.L. Biessen
KONINKLIJKE BIBLIOTHEEK, DEN HAAG Fluiter, Kees
The selective uptake of high density lipoprotein cholesterol esters by the liver: The role of scavenger receptor in reverse cholesterol transport / Kees Fluiter
Proefschrift Leiden. - Met lit. opg. ISBN 90-74538-33-9
The studies presented in this thesis were performed at the division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research (LACDR), University of Leiden, The Netherlands.
The studies presented in this thesis were supported by the Netherlands Organisation for Scientific Research, counsil for Medical Research, and the Netherlands Heart Foundation, Medical Sciences Grant no. 902-523-096.
Financial support by the Netherlands Organisation for Scientific Research and the Netherlands Heart Foundation for the publication of this thesis is gratefully acknowledged. The printing of this thesis was further financially supported by:
Cover: cell triplet, photo by Kees Fluiter
Chapter 1 General introduction
1.1 Atherosclerosis and cholesterol 1.2 Lipoproteins
1.3 HDL metabolism 1.4 Familial HDL disorders
1.5 The anti-atherosclerotic properties of HDL Reverse cholesterol
1.5.2 activity of HDL 1.5.3 Anti-inflammatory effects of HDL 1.6 HDL
1.6.1 Scavenger receptor class B, type I 1.7 Scope of this thesis
Chapter 2 Scavenger receptor BI (SR-BI) substrates inhibit 21
the uptake of HDL cholesteryl esters
by rat liver cells.
Kees Fluiter and Theo J.C. van Berkel (1997), J. 326, 515-519
Chapter 3 In vivo regulation of scavenger receptor 33
and the selective uptake of high density lipoprotein
cholesterol esters in rat liver and
cells.
Kees Deneys R. van der Westhuijzen, and Theo J.C. van Berkel (1998), J. 273, 8434-8438
Chapter 4 Increased selective uptake in vivo and in vitro of 47
oxidized cholesteryl esters from high-density lipoprotein
by rat liver cells.
Kees Fluiter, Helene Eric Biessen, Gert M. Kostner,
Theo J.C. van Berkel, and Wolfgang Sattler (1996), Biochem. J. 319, 471-476
Chapter 5 Scavenger receptor BI mediates the selective uptake 61
of oxidized cholesterol esters by rat liver. Kees Fluiter, Wolfgang Sattler, Deneys R. van der Westhuijzen, and Theo J.C. van Berkel (1998), submitted
Chapter 6 General discussion and perspectives 77
Chapter 7 Summary 85
Chapter 8 Samenvatting voor 89
General Introduction
Chapter
l
1.1 Atherosclerosis and cholesterol
Cardiovascular disease (CVD) is the primary cause of death in the western world. Since 1918 CVD has been the No. 1 killer in the USA CVD claims more lives each year than next 7 leading causes of death combined. In the U.S.A. more than 41% of all deaths are caused by CVD, meaning an average death by CVD every 33 seconds. The mortality rate by CVD is almost similar in the Netherlands. According to the Global Burden of Disease study, ischémie heart disease will become primary cause of death for the world by the year 2020 Atherosclerosis, the scientific name of lesion formation in the arteries, which ultimately may result in a total blockade of arteries is accompanied by an inflammation reaction. The immune system responds to small injuries or a local dysfunction in the vessel wall, but sometimes the inflammation reaction is to causing clinical problems. The response to injury theory [3,4] is now generally accepted. In general, atherosclerotic process consists of three stages: the fatty streak, the intermediate lesion and the fibrous plaque lesion). Atherosclerotic lesions are characterized by an excessive deposition of cholesterol in the arterial wall, cell proliferation and cell accumulation. Subsequently this leads to reduction of the vessel lumen The initial reaction to physical damage of the lining of the vessels is the binding of monocytes and to the endothelial cells, by a migration of these cells to the underlying smooth muscle cells. The monocytes differentiate into macrophages which start to accumulate lipids and foam cells. Smooth muscle also begin to migrate and proliferate, and a sticky matrix of elastic fibres, collagen and proteoglycans is formed. The cells in the lesion form a communicative network, secreting a complex array of cytokines, growth factors and signalling molecules (reviewed by Ross which attract more cells into the lesion. Rupture of the lesion and thrombus formation at lesion site can cause occlusion of the arteries, leading to heart attacks and strokes.
General Introduction
1.2
In higher organisms, is transported through the blood plasma by lipoproteins. Lipoproteins are water-soluble high-molecular weight complexes, composed of a hydrophobic core and a hydrophilic shell. The core contains triglycérides and cholesterol esters, while the shell consists of a monolayer of cholesterol and one or several apolipoproteins. The apolipoproteins stabilize the particles, function as ligands for receptors, and act as for enzymes which modulate lipoprotein metabolism (reviewed in Four important lipoprotein classes can be distinguished based on their size and composition (table 1). The two major cholesterol carrying classes are low density lipoproteins (LDL) and high density lipoproteins (HDL). In humans, LDL is transporting the bulk of cholesterol through the body. High LDL levels in plasma appear to be positively correlated with the incidence of CVD
However, it is important to note that not all mammals use LDL as their principle transport system. Roughly, all mammals can be divided in either "HDL" or "LDL" mammals, depending on which lipoprotein is carrying mostly cholesterol. In contrast to LDL, HDL plasma levels are inversely correlated with the incidence of CVD, as already noticed by Gofman et al. in 1950 Since this observation, it is assumed that HDL protects against the development of atherosclerosis.
Table 1. Physical properties and composition of human plasma lipoproteins
LDL HDL density protein triacylglycerol phospholipids ester free cholesterol apolipoproteins <0.96 75-1200 1-2 80-95 3-6 2-4 1-3 0.96-1.006 30-80 6-10 45-65 5-20 6-22 4-8 1.019-1.063 19-25 18-22 4-8 18-24 45-50 6-8 B100 1.063-1.210 5-12 45-55 2-7 26-32 5-20 3-5
The values given for protein, triacylglycerol, phospholipid, cholesterol ester, and free cholesterol are expressed as percentage of total weight.
These VLDL remnants can be taken up by remnant receptors, but can also function as precursor for LDL. LDL is the major cholesterol carrier in man. LDL uptake is mediated by the LDL-(Apo [7], which is under strong feedback regulation by the cholesterol content of the cell. During circulation LDL can become modified either by oxidation or glycation Modified LDL is up by scavenger receptors (reviewed in which are part of the innate immune system. These scavenger receptors are not under any feedback regulation and allow macrophages to take up excessive amounts of cholesterol. This can result in the formation of foam cells in the atherosclerotic lesion. This also explains in part why high LDL levels are correlated with the incidence of CVD. Chylomicrons, VLDL and (modified) LDL are all routed to the lysosomal degradation pathway In contrast to the other lipoproteins HDL is not readily taken up and routed to the lysosomes. In addition, HDL is also the only
which is negatively correlated with the incidence of CVD. The metabolism of HDL and its function as carrier of cholesterol to the liver will be the focus of this thesis.
exogenous endogenous
LPL LPL LPL LPL LPL
capillary beds
General Introduction
1.3 HDL metabolism
HDL is suggested to be involved in the removal of excess cholesterol from cells to liver. This process is known as reverse cholesterol transport HDL forms a
population of particles, and can be sub-divided into several subclasses based on size, density and composition yields two basic HDL subclasses, (1.063< density > 1.125 and (1.125< density >1.21 In addition HDL can be divided into subclasses based on their apolipoprotein composition. Two major subclasses can be distinguished way: HDL only containing and HDL containing both and HDL can also contain apo C's, and apoE depending on the size of the particle, further increasing the number of HDL subfractions. Non-denaturing two-dimensional gel electrophoresis has been used to fractionate HDL in HDL with and smaller-sized HDL fractions with pre ß-mobilily Figure 2 shows a scheme for the metabolism of HDL (extensively reviewed in [21] and De synthesized HDL is initially not much more than free apoA-I which has acquired some This very small (heavy) HDL particle with pre is a very potent absorber of cellular cholesterol. Castro and [24] showed that cellular cholesterol, that is primarily taken up by this pre HDL, is conversed to discoidal pre ß-2 HDL. It matures into a spherical particle after interaction with lecithin cholesterol
(LCAT) in the blood stream. Both the initial discoidal particle with electrophoretic pre ß mobility and the resulting spherical particle, are very efficient in promoting cholesterol These particles, which are part of the subclass, mature subsequently into larger particles after further uptake and of cholesterol. The presence of cholesterol ester transfer protein (CETP) activity further modulates the composition of the HDL
CETP mediates the transfer of cholesterol esters in exchange for triglycérides
Therefore, esters can either be delivered directly to the liver by HDL or transferred to LDL. Finally, enriched triglycérides or the apo E containing can be degraded by the action of LPL and hepatic lipase, resulting in free apo lipoproteins, which can start full life cycle again, or smaller sized particles which are also able to absorb
PERIPHERY
LIVER
FC • HDL (pr. 8) o HDL liver parenchymal LDL ABSORPTION FCcholesterol transport
BILE
Figure 2. Schematic
of the HDL metabolism. See text for details.
1.4 Familial disorders of HDL metabolism
HDL deficiencies are extremely rare. They arc mostly diagnosed because patients do have corneal opacities and sometimes subcutaneous accumulations as a result of a decreased level or the absence of plasma HDL. Most of these HDL deficiencies do not pose an increased risk of CVD for the patients. The most prevalent cause of HDL deficiency is familial LCAT deficiency. The disorder is inherited in an autosomal recessive mode. The complete absence of a functioning LCAT results in reduced HDL levels, and most of HDL is discoidal. Very spherical HDL is found, and the particle is always very small (~6nm) Fish eye disease, so called because patients have corneal opacities which make the eyes look like those of boiled fish, is also a form of LCAT deficiency. However, in fish eye disease LCAT is partially functional. Toward LDL and VLDL LCAT activity appears fairly normal, while HDL is no longer a preferred substrate. Fish eye disease also results in severely reduced HDL levels. However, both familial LCAT deficiency and fish eye disease do not lead to premature CVD.
General Introduction
point a single acid substitution Arg 173 to Cys, called Milano, resulted in strong decreased HDL levels in plasma. However, this mutation is suggested to offer significant protection against atherosclerosis Recombinant carrying this mutation is now even regarded as a potential therapeutic agent for atherosclerosis.
1.5 The anti-atherosclerotic properties of HDL
The well-documented anti-atherogenic effect of HDL is very likely the cumulative result of the different properties of HDL. In this section the three main (putative) mechanisms by which HDL protects against atherosclerosis will be discussed. The proposed anti-atherosclerotic mechanisms of HDL are:
1- reverse cholesterol transport. 2- anti-oxidant activity
3- inhibition of cell adhesion during inflammation 1.5.1 reverse cholesterol transport
The classical explanation for the anti-atherosclerotic properties of HDL is the so-called reverse cholesterol transport as originally proposed by Glomset in 1968 In this model HDL accepts cholesterol from cells and transports it to the liver for biliary secretion. cholesterol transport can be described into three separate stages. First, the efflux of cellular to HDL and its esterificalion. Second, transport of cholesterol esters the blood by HDL and the interaction with LDL. Third, the uptake of the cholesterol esters by the liver.
Cholesterol efflux
The initial phase of reverse transport is the efflux of cellular cholesterol to HDL. cholesterol is transferred from the plasma membrane to the HDL particle. Especially the small HDL particles with pre ß were shown to be potent promotors of cholesterol efflux
In the original concept by Glomset the enzyme acyltransferase
process which does not require metabolic energy or the involvement of receptors. The aqueous diffusion model has been further specified by et al [46], taking into account the presence of cholesterol-rich and cholesterol-poor membrane domains. It has become increasingly evident that cholesterol is not randomly distributed in membranes (reviewed in The presence of such cholesterol domains may affect the transfer kinetics of cholesterol. It is predicted that cholesterol transfer from rich domains is slower than from cholesterol-poor domains since the cholesterol molecules are less tightly packed in domains and require thus a lower energy for desorption
Although, the cholesterol efflux rate to cyclodextrin, an artificial,
cholesterol acceptor, was not cell type dependent [48], the cholesterol rate to HDL was shown to be cell type specific This cell type dependency of the cholesterol efflux rate to HDL indicates that a diffusion model alone is not sufficient to describe cholesterol efflux. Certain specialized cell types are able to internalize HDL temporarily to facilitate cholesterol transfer to HDL, after which the HDL particle is resecreted by the cells. However, this process, known as retroendocytosis, is limited to specific cell types under specific conditions and is believed not to be of great physiological importance Many authors have suggested that a specific HDL-receptor is involved in reverse cholesterol transport. It was found that binding of HDL to certain cell types could actively promote translocation of cholesterol from intracellular cholesterol pools following a protein kinase C signalling pathway many HDL binding proteins have been identified [55-60], but except for one, none of these were shown to mediate cholesterol transfer between HDL and cells. Scavenger receptor BI (SR-BI) is up till now the only receptor proven to be able to mediate cholesterol transport between cells and HDL (reviewed in The role of SR-BI will be described in further detail in a later paragraph. Transport through the circulation
The route that is taken for reverse cholesterol transport after the uptake and esterification of cholesterol from cells by HDL, is dependent on the presence of cholesteryl ester transfer protein (CETP) CETP enables the transfer of cholesteryl esters from HDL to LDL and VLDL in exchange for triglycérides as reviewed by Lagrost The direction of this transport is in part regulated by the presence of the transfer inhibitory protein CETP can redirect part of the HDL cholesterol esters to LDL and VLDL. These cholesterol esters can then be delivered to the liver via the LDL/apo E receptor pathway or back to the cxtrahepatic For this reason CETP can exert an effect on atherosclerosis. Recent studies have shown that mice over-expressing human CETP increase their pre ß-HDL levels, which are potent initiators of cholesterol efflux Furthermore, preliminary studies by the group of
General Introduction
ester uptake by the liver
The final step in the reverse cholesterol pathway is the delivery of cholesterol esters to the liver. Depending on the activity of CETP a part of the cholesterol esters may be delivered to the liver via LDL/apo E receptor pathway. Binding of LDL to its receptor results in the classical endosomal uptake and lysosomal degradation pathway as was described by Goldstein and Brown However, cholesterol ester delivery to the liver by HDL occurs without uptake of the HDL particle. In HDL turnover studies, Glass and Pitmann [70,71] showed that the uptake of undegradable cholesterol ethers was higher than for tyramine cellobiose iodinated HDL, indicating that HDL esters can be taken up selectively without parallel uptake of the whole particle. Different cultured cells, i.e. liver cells, and macrophages of mouse and human origin [72,73] do express the selective uptake route. Selective uptake of HDL cholesterol esters could also be exerted by isolated plasma membranes of liver parenchymal cells and HepG2 cells In vivo however, selective uptake is a cell type specific process.
Pieters et al. [75] showed that within the liver only the parenchymal cells are responsible for the selective uptake of HDL cholesterol esters. Apart from the liver, only organs involved in steroid synthesis exhibit selective uptake of HDL cholesterol esters, especially both the adrenal cells and ovary cells The selective uptake of esters by the liver parenchymal cells is efficiently coupled to bile acid synthesis and secretion. When compared to LDL cholesterol esters, the biliary secretion of bile acids derived from HDL-cholesterol esters is twice as fast Goldberg that in rabbits 70% of cholesterol delivery to the liver occurred via LDL and 30% via HDL Because CETP activity in rabbits is approximately four times higher as in humans, selective uptake is still believed to be of major importance in HDL-cholesterol clearance in humans. In addition, selective uptake of HDL cholesterol esters could be demonstrated in mice ovcrexpressing human More recently it was shown that the absolute delivery of HDL esters to liver was decreased in apoA-I knockout mice
1.5.2. The anti-oxidant activity of HDL LDL oxidation
modification of LDL (OxLDL) ultimately results in the recognition of the modified LDL particle by scavenger receptors, which arc a part of the innate immune system Unlike the LDL receptor, scavenger receptors are not regulated by the intracellular cholesterol content. Therefore, oxidation of LDL may result in the unbridled accumulation of lipids by macrophages and endothelial cells that express these scavenger receptors. Furthermore, OxLDL alters the gene expression of many growth regulatory molecules and cytokines in endothelial cells [84-87], it can be cytotoxic [88], and exerts effects on the vascular tone OxLDL is expected to induce lesions by several mechanisms. Lipid hydroperoxides are the first intermediates that are formed during the LDL oxidation cascade. Further down oxidation cascade, a wide variety of aldehydes, ketones and hydroxylipids arc formed. The aldehydes generated during oxidation of LDL can form adducts to lysine groups of apolipoprotein resulting in recognition by scavenger receptors
HDL and anti-oxidant
Several lines of evidence have suggested a role for HDL in the protection of LDL against oxidation. vitro studies have shown that co-incubation of LDL with HDL decreases and catalysed generation of LDL peroxides Also, it has been shown in vivo, that the level of plasma conjugated dienes and trienes were reduced by 30% up to 24 hours after a bolus injection of 200 mg human into rabbits In the same study a significant negative correlation between conjugated diene concentration and HDL plasma levels in humans was shown.
The mechanism by which HDL achieves its anti-oxidant action has been a matter of speculation. Probably, anti-oxidant mechanisms are operative at the same time. The best established anti-oxidant effect of HDL is by paraoxonase that is associated with HDL. Human paraoxonase is a glycoprotein of 43,000 Da molecular weight, and was, due to its detoxifying properties of nerve gases, originally in the focus of of toxicologists. Interest in paraoxonase in the context of atherosclerosis started by a report enzyme activity was substantial lower in CVD patients as compared to control subjects Later it was shown that paraoxonase had clear antioxidant activities. The of protection offered by HDL to LDL oxidation was directly related its paraoxonase activity et al. that HDL could scavenge for metal ions by HDL-associated ccruloplasmin and transferrin and thus prevent catalysis of lipid peroxidation However, in same studies, prepared HDL, which loses most of its associated still retained anti-oxidant activity. In addition, HDL decreased the capacity of OxLDL to form foam cells
General Introduction
hydroxides which is mediated by and The HDL specific reduction of hydroperoxides to hydroxides is accelerated in the presence of CETP [103] and is further increased by factors secreted by the liver Sattler and Stocker showed that oxidized cholesterol esters in HDL are taken up to a greater extent by HepG2 cells than native cholesterol esters This suggested that oxidized lipids, after transfer to HDL, can be rapidly detoxified. The studies presented in this thesis (chapters 4 and 5) focus on whether the liver can take the oxidized lipids from HDL and thus contribute to the protection against LDL oxidation and consequently atherosclerosis.
1.5.3 The anti inflammatory effects of HDL
HDL plasma levels are inversely correlated with the incidence of atherosclerosis. Since during atherosclerosis an inflammatory response is involved, the last couple of years more attention has been given to the potential anti-inflammatory properties of HDL. Two types of HDL mediated anti-inflammatory actions can be distinguished. The first is the consequence of the previously atherosclerotic mechanisms. Especially the link between anti-oxidation mechanisms and anti-inflammation is apparent. By removing biological active oxidized lipids from modified LDL, activation of cells in the artery wall and monocytic transmigration may be It was also recently found that HDL from transgenic mice over-expressing induced monocyte transmigration through cells of the artery wall. This
response was associated with a decreased content of paraoxonase activity However, there appears to be also a second anti-inflammatory property to HDL. HDL was repeatedly shown to inhibit cytokine-induced expression of endothelial cell adhesion molecules Physiological concentrations of HDL were shown to inhibit tumour necrosis factor-alpha or mediated induction of the leucocyte adhesion molecules, vascular cell adhesion intercellular adhesion
1.6 HDL receptors
The search for a functional HDL receptor has taken various decades. Although during these years a number of HDL binding proteins have been described, a serious candidate protein was described only recently. Several groups have HDL binding proteins by blotting in extrahepatic and hepatic cells The molecular weight of these
proteins is between 80 kD and 180 kD. Most HDL-binding proteins seem to be related to a 110 kD protein which was cloned and isolated by the group of This HDL-binding protein is up regulated after cholesterol loading of cells It possesses a very peculiar structure as the gene consists of 14 imperfect tandem repeats each coding for approximately 70 acids. Each tandem repeat contains two amphipalic helices This protein does not posses a classic hydrophobic membrane-spanning domain, and a potential function as HDL receptor seems questionable. In the liver of several species two HDL-binding proteins were identified which differ in binding characteristics The two HDL-binding proteins that were isolated by the group of Fidge appear to be distinct from the HDL-binding protein that was cloned by the group of Oram. The HDL-binding as isolated by Fidge et al. [57] are glycosylated and do contain a hydrophobic membrane spanning domain, a cytoplasmic sequence and an extracellular receptor domain. However, receptors were not shown to exert selective uptake of esters.
1.6.1
Scavenger receptor class B, type I.
The most characteristic of HDL metabolism is the property of selective cholesterol ester uptake without concomitant particle uptake. The only receptor which is able to mediate such selective transfer, surprisingly turns out to be a member of scavenger receptor gene family.
scavenger receptor gene family
General Introduction
SR-C
Figure 3. Structures of the class A, class B, and class C scavenger receptors SR-AI and SR-AII are generated by splicing of transcripts of the same gene. They consist of transmembrane, spacer and collagenous domains. The collagenous domain binding. SR-AI contains a carboxy-terminal scavenger receptor cyslein rich (SRCR) domain. The class B receptors are predicted to have Iransmembrane and a single large extracellular loop, that a set of conserved (indicated) and putative glycosylation sites SR-C comprises several domains, a small cytoplasmic domain (hatched), a transmembrane domain (black) followed by a small spacer domain, a rich like domain which may be heavily glycosylated (boxed), a
domain (open) followed by another spacer domain (black), a large MAM domain (striped), and at the there are finally complement control protein (ccp) domains (black)
class B
The class B receptors, includes two plasma membrane proteins, CD36 [115] and
and also a integral membrane protein (LIMPII), [117] and a drosophila membrane protein The members of the scavenger receptor class B receptors share a acid sequence identity and are predicted to have two transmembrane segments. most conserved region of the class B scavenger receptors is a domain (luminal in the case for LIMPII) that contains similarly spaced residues and sites for sugar attachments. Recently it was found that variant of SR-BI, the result of RNA splicing, are present in mice. These splice forms differ completely in the putative cytoplasmic domain of this receptor SR-BI and its human homolog CLA-1 suddenly came into focus when it was discovered that SR-BI not characteristically for scavenger receptors, could bind modified LDL but also native lipoproteins with high affinity, especially HDL
scavenger receptor had a 85% with the human which function until this discovery was not determined like CD36, is palmitoylated and was found to reside in specialized membrane domains on the cell-surface, the caveolae Both SR-BI and CD36 bind modified lipoproteins, but unlike members of the class A scavenger receptors the binding of these ligands is not inhibited by and acid phospholipids are also a substrate for SR-BI [124], which suggested that it may play a role in the clearance of apoptotic cells, but unlike any other scavenger receptor SR-BI binds native LDL and HDL with high affinity.
SR-BI became a serious contender to be a HDL receptor when it was demonstrated that it mediated selective transport of lipids. Chinese hamster ovary cells with SR-BI not only displayed HDL binding with a high affinity and saturability but were also able to selectively take up HDL cholesterol esters Furthermore, SR-BI and its human counterpart CLA-1 are expressed at high levels in the adrenals, ovary, and liver These organs are classically known for their ability to mediate selective uptake of HDL cholesterol esters Adenoviral overexpression of SR-BI in mouse liver provided further evidence that SR-BI may function as a HDL receptor. Overexpression of SR-BI resulted in a reduction in HDL cholesterol levels in plasma The same study also indicated that SR-BI can be present in the membranes of parenchymal cells, tentatively a possible role of SR-BI in bile transport. Krieger and coworkers also a SR-BI knock-out mouse, which had increased plasma cholesterol levels and significant larger HDL particles, indicating that reverse cholesterol transport was hampered
SR-BI is not only suggested to mediate selective uptake of esters but also may be involved in the efflux of cholesterol from extrahepatic cells to HDL. six different cell types, including macrophages, the rate of cholesterol efflux was correlated with the cellular SR-BI expression In fact, SR-BI is now thought to mediate lipid transport in general between HDL and cells. Also it now becomes clear that selective cholesterol ester transport from HDL is always coupled to transport of phospholipids, which may shield the cholesterol esters from having to traverse the aqueous phase between HDL and the cell plasma membrane
SR-BI tissue expression is regulated by the cellular status. Adrenal SR-BI expression is up-regulated in hepatic lipase, and LCAT knockout mice All three knock-outs have a dysfunctional HDL cholesterol ester delivery to the adrenals and as a result the adrenal cells have severely depleted cholesterol stores. SR-BI tissue expression in rats was also found to be regulated by estradiol treatment. High-dose treatment reduced SR-BI in the liver and increased SR-SR-BI in the adrenals and corpus luteal cells of the ovary, while human chorionic gonadotropin administration induced an increased SR-BI expression in the cells of the testis Recently it was shown that in the adrenals is under transcriptional control by the transcription factor, steroidogenic factor 1 The regulatory sequences of SR-BI/CLA-1 in the liver, which react to estradiol treatment, are still not known.
General Introduction
1.7 Scope of this thesis
This thesis focuses on the role of in the selective removal of native and oxidized cholesterol esters from HDL by the different liver cells. The selective uptake of cholesterol esters forms the final step in reverse cholesterol transport. The role of SR-BI in uptake of HDL cholesterol esters by steroidogenic tissues has been intensively studied by others. However, the role of SR-BI in the hepatic uptake of HDL cholesterol esters in vivo is not yet fully examined. Especially, the expression pattern of SR-BI in different liver cells and the regulation of SR-BI in these cells is investigated and correlated to the selective uptake of HDL cholesterol esters. In we paid special attention to the role of the liver and SR-BI to one of the proposed mechanisms which is mediated by HDL. It will be described the protection offered by HDL against atherosclerosis can be the result of several mechanisms working cooperatively.
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substrates inhibit selective uptake of HDL-CE
Chapter
2
Scavenger receptor (SR-BI) substrates inhibit the selective uptake of HDL cholesteryl esters by rat liver cells.
Abstract
HDL cholesteryl esters (HDL-CE) are selectively taken up by liver parenchymal cells without parallel uptake, and this selective uptake route forms an important step in reverse cholesterol transport. Recent data from Acton et al. Science 271, 518-520]
provide evidence that scavenger receptor B can mediate selective uptake of HDL-CE. In order to identify if selective uptake of by rat liver parenchymal cells can be mediated by a protein with scavenger receptor properties we performed in vitro competition experiments with substrates for scavenger receptors. Addition of either LDL, acetylated LDL (AcLDL) or oxidized LDL (OxLDL) did only decrease the association of HDL particles to parenchymal cells as measured by association was inhibited by AcLDL for up to 35%, while addition of OxLDL did inhibit HDL-CE association for thereby completely blocking the selective uptake of HDL-CE. Visualisation studies with HDL, labelled with a fluorescent cholesteryl ester analog, confirmed the OxLDL mediated complete inhibition of selective uptake by rat liver parenchymal cells. The inhibition of selective uptake by OxLDL was insensitive to the additional presence of acid (poly I), indicating that the inhibitory effect does not involve a poly I sensitive site. phospholipid liposomes inhibited HDL-CE association for 40%, neutral liposomes were ineffective. The inhibition of the uptake of in liver parenchymal cells by modified LDL, in particular OxLDL, and anionic suggests that, in liver scavenger receptor is responsible for the efficient uptake of HDL-CE.
Introduction
High-density lipoproteins (HDL) may exert the established anti-atherogenic effects by various mechanisms [2,3J. Reverse cholesterol transport, as first proposed by Glomset [4], is the best established mechanism of action. In this concept HDL accepts excessive cholesterol from exlrahepatic cells for transport to the liver Peripheral is primarily accepted by small HDL particles with pre-ß mobility After esterification by lecithin-cholesterol acyltransferase the HDL cholesteryl esters are delivered to liver either directly or by LDL as a consequence of the action of cholesteryl ester transfer protein (CETP). The direct uptake route of HDL cholesteryl esters (HDL-CE) by liver parenchymal cells is characterized by the selective uptake of the cholesteryl without simultaneous uptake of the holoparticle
The selective uptake route is efficiently coupled to bile acid formation and secretion The mechanism of selective uptake of HDL-CE is largely uncstablished. It is restricted to the adrenals, ovary, and liver [7,9], while within the liver the parenchymal cells are solely responsible for the uptake of HDL-CE Rinninger et al. [10] showed that there are distinct sites on liver parenchymal cells for the binding of the protein moiety of HDL and
CE uptake.
substrates inhibit selective uptake of
including both modified lipoproteins, native lipoproteins and also anionic phospholipids However, scavenger class B receptors do not bind the broad array of polyanions (e.g. fucoidin,
acid) which are classical ligands for scavenger class A receptors. In vivo SR-BI is expressed mainly in the adrenals, ovary and to a much lesser extent in the liver of rats and mice [15, In the present work the potential role of SR-BI in the selective uptake of HDL-CE by rat liver is investigated.
Previous studies from our group provided evidence for the presence of a lipoprotein binding site on rat liver parenchymal cells binding multiple types of lipoprotein It was also shown that rat liver parenchymal cells did contain a high affinity binding site for AcLDL and OxLDL that is insensitive to poly-inosinic acid
In study we investigated the potential role of scavenger receptor in the selective HDL-CE uptake by rat liver parenchymal cells, by performing in vitro competition studies with ligands specific for scavenger receptor B.
Experimental
Materials
oleate and (carrier free) in NaOH were obtained from (Little Buckinghamshire, England). (NBD) linoleate was obtained from Molecular Probes (Eugene, Oregon, U.S.A.). Egg yolk was obtained from (Buchs, the PL phospholipids kit, the cholesterol oxidase-peroxidase aminophenazone kit, and the glycerolphosphate oxidase-peroxidase aminophenazone (GPO-PAP) kit was from Boehringer Mannheim (Mannheim, Germany). Ethylmercurithiosalicylate (thimerosal), serum albumin (BSA, fraction V), and collagenase type I and type IV were from Sigma (St. Louis, MO, USA). Dulbecco's modified Eagle medium (DMEM) was from (Irvine, Scotland). All other chemicals were of analytical grade.
Animals
Throughout study male Wistar WU rats were used (200-250 g); They had free access to food and water. Prior to the experiments, the rats were anaesthetized with Nembutal given
liposome preparation
liposomes were obtained by sonication of egg yolk
in a molar ratio of The lipids were mixed in chloroform and dried under a stream of Sonication was carried out with an soniprep 150 for 40 minutes (amplitude 12 at 52°C under a constant stream of argon in a M KC1 / 10
/ 1 EDTA / 0.025% buffer, pH 8.0. particles with a density of 1.03 were isolated by density gradient ultracentrifugation and dialysed against phosphate buffered saline (PBS) / EDTA. The liposomes were stored at 4°C under argon. The phospholipid content the particles was measured by an enzymatic colorimetric assay. The average diameter of the was determined by photon correlation spectroscopy, (System 4700 C, Malvern
Isolation and labelling of lipoproteins
Human HDL and LDL were isolated from the blood of healthy volunteers by differential ultracentrifugation as described by Redgrave et al. HDL and LDL were dialysed against EDTA. HDL was labelled with or cholesteryl linoleate by exchange from donor particles as reported previously The specific activity of the labelled
HDL varied between HDL and for varied
50000-100000 The labelled HDL was dialysed against EDTA and passed through a heparin-Sepharose affinity column to remove apo E-containing particles Routinely the HDL fraction was checked for the absence of apo E by 10% followed by Coomassie Blue staining. After the labelling procedure the radiolabelled HDL was checked for hydrolysis of the cholesteryl ester labels by a & Dyer extraction [21] followed by thin layer chromatography. Hydrolysis of the cholesteryl ester was always less than 5%. The effect of the labelling procedure on the composition of HDL was analyzed by measurement of
cholesterol, cholesteryl ester, and triglycéride content (with the PL kit, CHOD-PAP kit and GPO-PAP kit, respectively). The density, electrophoretic ex-mobility and particle size (photon correlation System 4700 C, Instruments) were also labelled HDL was only used when there was no change observed in the measured composition or physical characteristics as compared to the original unlabelled HDL. HDL was iodinated by the
method of McFarlane [22] as modified by Bilheimer et al
Before use LDL was dialysed against PBS 10 EDTA. Acetylalion of LDL was performed by acetic anhydride as described LDL was oxidized by exposure to as described in detail earlier
In vitro studies with freshly isolated rat hepatocytes
Parenchymal liver cells were isolated by perfusion of the livers of male Wistar WU rats (200-250 g) with collagenase at 37°C as described The viability (>95%) of the obtained parenchymal cells was checked by trypan blue exclusion. The cells from the last centrifugation step were resuspended in oxygenated DMEM supplemented with 2% BSA, pH 7.4. For competition studies 1-2 of parenchymal cell protein was incubated with different amounts of protein of unlabelled human HDL, LDL, AcLDL, OxLDL, or neutral and anionic liposomes in the presence of an indicated amount of radiolabelled HDL for the indicated periods in 1 ml DMEM containing 2% BSA at 37°C. Cell incubations were performed in a circulating lab shaker (Adolf Kühner AG, Switzerland) at 150 Every hour the incubations were briefly oxygenated. The viability of the parenchymal cells remained higher than 88% during these long term incubations [24] . After incubation the cells were for 2 min at 600 rpm in an Eppendorf centrifuge and washed 2 times in 50 mM 0.15 M BSA, pH 7.4 at 4°C. Subsequently, the cell pellet was washed in a similar medium without BSA. cells were in N and the protein content and radioactivity were determined.
Confocal microscopy
substrates inhibit selective uptake of HDL-CE
objective which was equipped with a confocal visualisation system U.K.). The microscope was fitted with a incubation chamber to allow incubation of the cells at
Protein determination
Protein was determined according to et al. [25] with BSA as standard.
Results
Effect of modified lipoproteins on the selective uptake of HDL cholesterol esters.
Freshly isolated rat liver parenchymal cells were incubated for three hours at 37°C with HDL, either iodinated or labelled with At this time point the association of
labelled HDL (202±14 ng cell protein) apparently exceeds (36±3 ng cell protein) association 5.6 times. As by Pittman et al. [7] data of cholesteryl ester association are expressed in terms of apparent particle uptake i.e. the amount of apparent HDL protein taken up is calculated from the amount of CE tracer associated with cells. Therefore, the amount of radioactive CE tracer associated to the cells is expressed as apparent HDL protein association, as calculated using the specific activity of the labelled HDL. The ability of (modified) lipoproteins to compete for and association was tested by co-incubation with either 100 of protein unlabelled HDL, LDL, AcLDL or OxLDL (figure 1). Both HDL particle association, as measured by association, and
HDL association were inhibited for approximately 50% when 100 unlabelled apo HDL protein was added. Further inhibition of both association and HDL association up to 75% could be achieved by increasing the excess of unlabelled HDL to 500 (data not shown). In contrast to HDL, addition of either 100 of LDL protein or modified LDL protein did only marginally (<10%) decrease the cell association of
HDL association was approximately 20% decreased by addition of LDL. However, addition of AcLDL led to a significant inhibition of 35 % while OxLDL decreased
control HDL LDL
Figure 1. of native and modified lipoproteins on the cell association of l| or labelled HDL. liver parenchymal cells were incubaled for 3 hours at 37°C with 10 labelled HDL in the absence or presence of 100 ug protein of unlabelled competing lipoproteins in 2% BSA. The value for association of labelled HDL was 202±14 ng cell protein and for association was 36±3 ng cell The association is expressed as the percentage of the radioactivity obtained in the absence of competitor. The results are given as means ± S.E.M. (n=3 separate cell isolations). * indicates very significant difference between the value and the control; p<0.005. ** indicates extremely significant difference
the value and the control; (unpaired student's t-test).
The possibility of exchange of to the other lipoproteins was tested by isolating the lipoproteins after incubation for 3 hours at Both gradient density ultracentrifugation, and agarose gel electrophoresis were performed. Less than 5% of the was in the LDL, AcLDL, or OxLDL fraction after incubation for three hours at 37°C, that exchange to competitors could not explain the achieved results.
Effect of OxLDL on selective HDL-CE uptake.
The inhibitory effect of OxLDL on the selective uptake of HDL-CE was further
substrates inhibit selective uptake of HDL-CE
B
15020 40 60 80 20 40 80
(ug/ml)
Figure 2. of concentrations of OxLDL and poly I on the cell association of (A) or labelled HDL. Rat liver cells were incubated for 3 hours at with 10 labelled HDL in the presence of the indicated amounts of OxLDL and when indicated poly I (100 The
is expressed as the percentage of the radioactivity obtained in the absence of competitor. The 100% value for association of labelled HDL without competitor was 202±14 ng cell protein and for
without was 36±3 ng cell protein. The results are given as means ± S.E.M. cell
uptake of HDL-CE was also followed with confocal laser scanning using HDL labelled with a fluorescent cholesteryl ester analog. The uptake of fluorescent linoleate was followed up to three hours of incubation with Parenchymal cells. After 10 minutes at 37°C the label was mainly associated with the plasma (fig. 3). During the incubation period some parenchymal cell-pairs, which were not separated during isolation procedure regained their cellular polarity and formed active bile These so-called parenchymal cell couplets [26] allow to follow bile-directed transport fluorescent linoleale. At three hours of incubation at 37°C the label inside the cell was concentrated in at the apical side of the parenchymal cell couplets, near bile canaliculus. Addition of 100 OxLDL protein abolished uptake of fluorescent
Figure 3. Visualisation of the interaction of NBD cholesteryl linoleate labelled HUL with liver cell couplets and the effect of OxLDL. Freshly parenchymal cells were pre-incubated on glass
for two hours at 4°C in supplemented with 2% and NBD cholesteryl linoleate labelled HDL in the presence or absence of 100 OxLDL. Cells were analysed with a confocal microscope fitted with a incubation chamber to allow incubation of the cells at 37°C. The fluorescence of NBD cholesteryl linoleate was followed at 37°C in the absence of OxLDL for 10 minutes (left) or 120 minutes (middle) and in the presence of OxLDL for 120 minutes (right).
Selective uptake is sensitive for anionic phospholipids.
substrates inhibit selective uptake of HDL-CE
Both inhibited the characteristic fast initial association of HDL-CE by liver parenchymal cells indicating that inhibition of cellular HDL-CE association was caused by blocking the initial cellular association mechanism of HDL-CE and not by exchange to the inhibitors. Control PS-Lipo 1(X) ± 30 100 100 ± 20 58.3 ± 8.7*
1. of phosphatidyl and neutral liposomes on the cell association of or labelled HDL. Rat liver parenchymal cells were incubated for 3 hours at 37°C labelled HDL in the or presence of liposomes (100 phospholipid in with 2% BSA. The 100% value for association of labelled HDL was 202±14 ng cell protein and for association was 36±3 cell protein. The association is expressed as the percentage of the radioactivity obtained in the absence of competitor. The results are given as means ± (n=3 separate cell indicates significant difference between the value and the control; p<0.005 (unpaired student's t-test).
4. Time course of the cell-association of labelled by rat liver parenchymal cells and the of or phosphatidyl serine liposomes. Rat liver parenchymal cells were incubated for the indicated
Discussion
Selective delivery of CE from HDL to the liver is an important direct route for reverse cholesterol transport. The precise mechanism of this uptake and the mediators is not known. Scavenger receptor might be involved in this process [1], but until now direct involvement of in reverse cholesterol transport has not been shown. Unlike scavenger class A receptors, SR-BI is insensitive for like poly I The presence of a scavenger receptor on rat liver parenchymal cells which is not to poly I, was already shown earlier by van Berkel et al [18], while also direct competition of LDL binding to parenchymal cells by HDL was observed However, the association of particles to rat liver parenchymal cells was only effectively competed for by HDL itself, while the maximal competition by LDL, AcLDL and was not significant. In contrast, the
uptake of HDL-CE by liver parenchymal cells is completely blocked by the of OxLDL, while also anionic and to a lesser extent AcLDL are effective inhibitors. It can be that OxLDL inhibits the uptake of HDL-CE nearly completely, as the residual association of HDL-CE equals particle association as measured with It thus appears that the selective HDL-CE uptake by rat liver parenchymal cells is mediated by a recognition site which possesses recognition properties characteristic for scavenger receptor The finding that the inhibitory effect of OxLDL on HDL-CE was not influenced by the simultaneous presence of poly I is consistent with the characteristic property of scavenger receptor BI, which the other scavenger receptor classes, is insensitive to poly I Both liposomes containing anionic phospholipids and poly I increase in association. This might be by an increase in aspecific association mediated by the charge of poly I and anionic phospholipids, acting as a bridge between cells and particles. HDL-CE association is increased similarly in the presence of poly I. However, poly I, the anionic phospholipids inhibit association of HDL-CE for almost 50%. When taken into account the increase in HDL association mediated by anionic phospholipids, it can that like OxLDL, anionic phospholipids completely block selective uptake of HDL-CE.
The question arises why the total HDL association is only marginally affected by the inhibitors of selective HDL-CE uptake like OxLDL, the selective CE-uptakc. However, it can be calculated, using our earlier data on amount of poly I insensitive binding sites for OxLDL on liver parenchymal cells [18,28] these sites contribute only up to 1% of all HDL binding sites. So it appears that selective uptake site of HDL-CE only to a low extent to total amount of HDL binding sites, suggesting that its coupling to
is highly efficient. It also may seemingly HDL-CE uptake is not related to HDL particle binding as observed previously Since HDL-CE association exceeded HDL association 5.6 times after incubation of 180 minutes, it can be calculated that HDL particles must 19 seconds at these
substrates inhibit selective uptake of HDL-CE
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Hepatic expression and selective uptake of HDL-CE
Chapter
3
In vivo regulation of receptor and the selective uptake of high density lipoprotein cholesterol esters in rat liver and
cells.
