High-density lipoproteins (HDLs) encompass a heterogenous class of lipoproteins that have in common a high density (>1.063 g/ml) and a small size (Stokes' diameter: 5 to 17 nm). The majority of the HDL particles contain apo A-I. Differences in the quantitative and qualitative content of lipids, apolipoproteins, and lipid transfer proteins result in the presence of various HDL subclasses that are characterized by differences in shape, density, size, charge, and antigenicity.62
HDL can be separated by ultracentrifugation into HDL2 (d = 1.063 to 1.125 g/ml) and HDL3 (1.125 to 1.21 g/ml).63 Nondenaturing polyacrylamide gradient gel electrophoresis (PAGGE) further discriminates by size HDL2 into HDL2b and HDL2a, as well as HDL3 into HDL3a, HDL3b, and HDL3c.64 Immunoaffinity chromatography fractionates HDL by apolipoprotein composition. The serial use of anti-apo A-I- and apo A-II-immunoaffinity-chromatography separates HDL into two subclasses, namely one particle that contains apo A-I and apo A-II (i.e., LpA-I/A-II), and a second particle containing apo A-I but no apo A-II (i.e., LpA-I).62,65,66
Following agarose gel electrophoresis of plasma and anti-apo A-I-immunoblotting, the majority of apo A-I is present in a fraction which migrates with α-electrophoretic mobility and is designated α-LpA-I, apoA-I-containing lipoprotein. This fraction eventually contains all of the cholesterol that is quantified in the routine laboratory as HDL-cholesterol. It consists of various particles that differ by size and density, as well as by the stoichiometric content of various apolipoproteins. Approximately 5 to 15 percent of apo A-I in human plasma is associated with particles that have electrophoretic pre-β-mobility and that can be further distinguished by subsequent PAGGE and anti-apo A-I-immunoblotting into pre-β1-LpA-I, pre-β2-LpA-I, and pre-β3-LpA-I (Fig. 122-1 A, C, and E).62,67– 72 Pre-β1-LpA-I are the smallest particles, are discoidal in shape, and contain apo A-I either as a lipid-free apolipoprotein or in association with a few molecules of sphingomyelin and phosphatidylcholine. Studies in which plasma was initially incubated with 3H-cholesterol-labeled fibroblasts followed by separation by two-dimensional PAGGE identified pre-β1-LpA-I as the predominant initial acceptor of cell-derived cholesterol. From this particle, cholesterol appears to be transferred first to pre-β2-LpA-I and then to pre-β3-LpA-I. The presence of lecithin:cholesterol-acyltransferase (LCAT) and cholesteryl ester transfer protein (CETP) enables this particle to esterify cell-derived cholesterol and transfer the cholesteryl esters to α-HDL.62,68,70– 73 The major fraction of cell-derived cholesterol, however, appears to be transferred to α-LpA-I without prior esterification in preβ3-LpA-I.70 The combination of agarose gel electrophoresis, PAGGE, and immunoblotting has facilitated the detection of other lipid-poor HDL subclasses that occur at relatively low plasma concentrations. These particles contain only apo E (γ-LpE) or apo A-IV (LpA-IV-1 and LpA-IV-2) as their only apolipoproteins, and serve, like pre-β1-LpA-I, as initial acceptors of cell-derived cholesterol into the plasma compartment.62,74– 77 It is also important to note that relative to the concentration of lipid-rich α-HDL the concentration of these lipid-poor particles is increased in extravasal compartments, including the lymph where reverse cholesterol transport is initiated in vivo.72,78– 80
Agarose gel electrophoresis (A,B,C,D) and two-dimensional nondenaturing polyacrylamide gradient gel electrophoresis (E,F) of plasma from a normolipidemic control (A,C,E) and a patient with Tangier disease (B,D,F). Lipoproteins were stained either with fat red (A,B) or anti-apo A-I antibodies (C,D,E,F). In normal plasma, the majority of apo A-I is found in a particle with electrophoretic α-mobility, and a minority in particles with electrophoretic pre-β-mobility (C,E). In Tangier plasma, apo A-I is only found in pre-β1-LpA-I (F). For details of the method see references60 and 67 to 70.
Lipid-rich α-HDLs arise from lipid-poor particles or from lipid-free apolipoproteins.62,71,72,81,82 These lipid-poor HDL-precursors are produced as nascent HDL by hepatocytes83– 88 and the intestinal mucosa,89– 91 dissociate from chylomicrons and VLDL during lipolysis,92– 94 or are generated by the remodeling of HDL by CETP,95– 99 phospholipid transfer protein (PLTP),100,101 and hepatic lipase (HL) 102 (Fig. 122-2). Nascent HDL of intestinal origin contain apo A-I and apo A-IV, and nascent HDL of hepatic origin contain apo A-I, apo A-II, and/or apo E. It is not yet known whether nascent HDL are assembled intracellularly in the endoplasmic reticulum or extracellularly by association of lipid-free apolipoproteins with phospholipids and cholesterol released from the plasma membrane. In agreement with the latter model, lipid-free apolipoproteins A-I, A-IV, and E promote efflux of phospholipids and cholesterol from cultured hepatocytes, macrophages, and fibroblasts, resulting in the formation of lipid-containing HDL-like particles.58,71,72,82,103– 108
Pathways involved in the generation and conversion of HDL. Mature HDL3 and HDL2 are generated from lipid-free apo A-I and lipid-poor pre-β1-HDL as precursors. These precursors are produced as nascent HDL by the liver or intestine or are released from lipolyzed VLDL and chylomicrons or by interconversion of HDL3 and HDL2. ABC1 may be involved in the initial lipidation of apo A-I into pre-β1-LpA-I (F). For details see text. ABC1 = ATP-binding cassette transporter 1; CETP = cholesteryl ester transfer protein; HL = hepatic lipase; LCAT = lecithin:cholesterol-acyltransferase; LPL = lipoprotein lipase; PLTP = phospholipid transfer protein; SR-BI = scavenger receptor BI.
These lipid-poor HDL precursors are converted into mature, lipid-rich, and spherical α-LpA-I by the combined effects exerted by the acquisition of phospholipids and unesterified cholesterol, the LCAT-mediated esterification of cholesterol, and the association of additional apolipoproteins.107,109– 114 In this process, plasma membranes of peripheral cells serve as donors of lipids, and apo B containing plasma lipoproteins, as well as their remnants, are a source of apolipoproteins and lipids.60,61,107,115 The initial products are small HDL3. The particles mature into HDL2 following esterification of cholesterol through LCAT81,116,117 and PLTP-mediated fusion of α-HDL3 particles118– 120 (Fig. 122-2).
Cholesteryl esters of α-HDL are removed from the circulation by two major direct pathways and one indirect pathway: (a) Hepatocytes and steroid hormone-producing cells express scavenger receptor BI (SR-BI), which binds HDL and mediates the selective uptake of cholesteryl esters without internalizing HDL proteins.121– 123 (b) A subpopulation of HDL containing apo E are internalized by hepatic apo E receptors.124– 127 (c) CETP exchanges cholesteryl esters of HDL2 with triglycerides of VLDL, IDL, and LDL, and the HDL-derived cholesteryl esters are removed from the circulation via the LDL-receptor pathway.128,129 As products of this process, smaller HDL3, as well as lipid-free apo A-I or pre-β1-LpA-I, are formed.95– 99 HDL3 and pre-β1-LpA-I are also generated by the hydrolysis of triglycerides and phospholipids of HDL2 by hepatic lipase (HL), which appears to serve as a coreceptor for HDL binding sites on hepatocytes.102,130
The interconversion of HDL 3 into HDL2 by PLTP, as well as the removal of lipids from HDL2 by SR-BI, CETP, and HL, regenerates pre-β1-LpA-I or lipid-free apo A-I.95– 102 These small lipoprotein particles can easily leave the plasma for the extravascular space.71,72,82 There it can serve as an acceptor of cellular lipids and thus, again, start the generation of HDL. However, in the kidney, these small particles are filtered and removed from the plasma.131 Cubilin, the receptor for intrinsic factor, mediates the reuptake of apo A-I from the lumen of the proximal tubule.132
Regulation of the Cellular Cholesterol Homeostasis
The compartmentalization of cholesterol within the cell is regulated.133– 135 The plasma membrane of mammalian cells accounts for 60 to 90 percent of cellular unesterified cholesterol, 95 percent of which is localized in the cytofacial leaflet of the bilayer membrane.133,135– 137 The lateral distribution of cholesterol within the plasma membrane is organized in microdomains. Coated pits, that is, clathrin-stabilized plasma membrane invaginations that contain lipoprotein receptors, have less cholesterol and sphingolipids than the rest of the plasma membrane. In contrast, caveolae, detergent-resistant plasma membrane invaginations, which are characterized by the presence of caveolins but the absence of clathrin, are enriched in both cholesterol and sphingolipids. Relatively small amounts of unesterified cholesterol are found in intracellular organelles except in those that communicate with the plasma membrane (endosomes, lysosomes and trans-Golgi network [TGN]).133,135,138 The TGN is an acceptor of newly synthesized cholesterol from the endoplasmic reticulum (ER) and exogenous cholesterol from endocytic vesicles, lysosomes, and caveolae,133– 139 and subsequently distributes cholesterol and phospholipids either in the form of detergent-resistant and caveolin-containing vesicles (rafts or cytolipoproteins),133,135,140,141 or as secretory detergent-soluble vesicles133,134,141– 143 (Table 122-1).
Table 122-1: Intracellular Transport Pathways of Cholesterol |Favorite Table|Download (.pdf) Table 122-1: Intracellular Transport Pathways of Cholesterol
| ||Cell type ||Metabolism ||Inhibitors ||Mechanism ||Involvement of Golgi apparatus/TGN |
|ER → plasma membrane ||Ubiquitous ||Membrane integrity, RCT ||Nocodazole, progesterone (brefeldin A)* ||Rafts, caveolins? ABC1? (vesicles)* ||Yes |
|Lysosome → plasma membrane ||Ubiquitous ||Membrane integrity, RCT ||NPC mutation, progesterone, imipramine, bafilomycin ||NPC1, caveolins? ||Possibly |
|Lysosome → ER ||Ubiquitous, especially in macrophages and hepatocytes ||ACAT bile acid synthesis ||NPC mutation, progesterone ||NPC1, caveolins? rafts? ||Probably |
|plasma membrane → ER ||Ubiquitous ||Membrane integrity, ACAT ||Progesterone, imipramine ||ABC1? ||Yes |
High levels of unesterified cholesterol are cytotoxic.144 Therefore, the concentration of cholesterol in the cell is precisely regulated by de novo synthesis, influx, efflux, esterification, and, in some specialized cells, by synthesis and secretion of lipoproteins, steroid hormones, bile acids, or vitamin D.
Endogenous cholesterol is synthesized through several intermediates from acetyl-CoA as the precursor. The rate-limiting step of cholesterol synthesis is the reduction of β-hydroxy, β-methylglutaryl CoA (HMG CoA) to mevalonate145 (see Chap. 120). Transfer of newly synthesized cholesterol from the ER to the plasma membrane is rapid (half-time: 10 min) and adenosine triphosphate (ATP)-dependent. The inhibition by amphiphilic drugs such as progesterone, as well as inhibitors of microtubule formation including nocodazole, suggests the involvement of a raft pathway.133,135,146 Consistent with this concept is the finding of biosynthetic cholesterol in caveolae.133,135,147,148 However, in lipid-enriched cells this transfer was also inhibited by brefeldin A or monensin, which indicates that at least under certain conditions secretory vesicles are involved in the transport of cholesterol from the Golgi apparatus to the plasma membrane.143,149
As a component of plasma lipoproteins, exogenous cholesterol can be internalized by specific lipoprotein receptors and directed to mature lysosomes via a series of clathrin-coated endocytic vesicles for subsequent degradation.150,151 Within the lysosomes, cholesteryl esters are hydrolyzed by acid lipase followed by incorporation of unesterified cholesterol into lysosomal membranes (see Chap. 142). The majority of lysosomal cholesterol, however, is transferred to the plasma membrane and then to the ER for reesterification by acylCoA:cholesterol-acyltransferase (ACAT) 133,152,153 (see Chap. 120), or in specialized cells, to other locations for the synthesis of bile acids, lipoproteins, or steroid hormones. Up to 30 percent of lysosomal cholesterol may even be directly transferred to the ER.133 The release of cholesterol from lysosomes and its subsequent translocation to other cellular cholesterol pools depends on regulated transfer mechanisms133,134 because export of lysosomal cholesterol is impaired in Niemann-Pick disease type C (NPC) (see Chap. 145),154,155 in a mutant hamster ovary cell line,156 and in the presence of amphiphilic drugs.157 Unesterified cholesterol in NPC cells or fibroblasts treated with amphiphiles accumulates in lysosomes, late endosomes, and the TGN, indicating that the latter organelle is involved in the transport of lysosomal cholesterol to the plasma membrane, probably by vesicle formation.133– 136,158,159
In addition to receptor-mediated pathways, cells selectively take up unesterified and esterified cholesterol from lipoproteins independent of their protein constituents. The HDL receptor SR-BI mediates the selective uptake of esterified cholesterol into hepatic and steroidogenic cells.121– 123 Cholesterol-binding proteins located within the caveolae, so-called caveolins, appear to be involved in the selective uptake of unesterified cholesterol from plasma lipoproteins into many cell types.70,135,160 Unesterified cholesterol is then directed from the plasma membrane to the TGN via endocytic vesicles.135,139,161
Both endogenous and exogenous cholesterol initiate a negative feedback mechanism. An increase in the concentration of intracellular cholesterol down-regulates the expression of the genes for HMG CoA reductase and the LDL receptor, preventing the overloading of cholesterol in many cells that cannot take up exogenous cholesterol independently of the LDL-receptor pathway145,150 (see Chap. 120). By contrast, the uptake of modified LDL, triglyceride-rich lipoproteins, and their remnants by class A scavenger receptors (SR-A), LDL-receptor-related protein (LRP), and the VLDL-receptor, as well as the phagocytic uptake of cholesterol into macrophages, is not regulated by the intracellular cholesterol concentration.151,162 Increasing levels of unesterified cholesterol in the plasma membrane stimulate esterification of cholesterol by ACAT in the ER.151,153 Although most of the esterified cholesterol is de novo synthesized, some is of lysosomal origin and transferred to the ER either directly or indirectly via the plasma membrane. The transfer of cholesterol from the plasma membrane to the ER depends on an intact cytoskeleton and is blocked by progesterone.133,134,164– 167 Cholesteryl esters formed by ACAT appear as cytosolic lipid droplets, which give lipid-laden macrophages their foamy appearance.153 Cytosolic cholesteryl esters can be hydrolyzed by neutral cholesteryl ester hydrolase (NCEH), which is activated by a cAMP-dependent protein kinase A.168,169 Cholesterol released by NCEH is transferred to the cell membrane where it can be transported to the ER for reesterification by ACAT.133,134,153 This cycle of cholesterol and cholesteryl esters between ACAT and NCEH is interrupted by the presence of extracellular cholesterol acceptors, such as HDL, which cause cholesterol efflux.170,171
Cholesterol efflux from cells is a central step in the regulation of the cellular cholesterol homeostasis and the result of unspecific and passive as well as specific and active processes (Fig. 122-3).71,82,137,172,173
Regulation of cholesterol efflux from cells. Cholesterol efflux by aqueous diffusion onto lipid-rich lipoproteins, albumin, phospholipid vesicles, or cyclodextrins is slow and not inhibited by brefeldin A or proteolysis of cells. Expression of SR-BI can facilitate this process. Efflux by lipid-free apolipoproteins or lipid-poor particles is fast and sensitive to proteolysis or treatment of cells with brefeldin A. It is defective in Tangier cells and therefore depends on ABC1. The intracellular mobilization of cholesterol for specific efflux appears to involve a signal transduction process.
Protein-free phospholipid vesicles, synthetic cyclodextrins, and trypsinized HDL mediate a slow and unsaturable cholesterol efflux from all cell types investigated thus far.137,173– 181 This form of cholesterol efflux is not prevented by partial proteolysis of plasma membranes or by preincubation of cells with inhibitors of intracellular vesicular transport such as brefeldin A.58,182,183 It does not involve specific interactions with cell-surface receptors or the specific activation of cellular transport processes, but simply reflects aqueous diffusion of cholesterol out of the plasma membrane onto acceptor molecules.137,173 Although the loss in cholesterol from the plasma membrane can be replenished, this form of cholesterol efflux has little effect in depleting cells of intracellularly stored cholesteryl esters.170,173,183– 185
By contrast, lipid-free apolipoproteins A-I, A-II, A-IV, Cs, and E, as well as amphipathic synthetic peptides without sequence homology to these apolipoproteins, cause an efflux of both phospholipids and cholesterol, which is fast, saturable, unidirectional, independent of LCAT, and efficient in reducing the content of cytosolic cholesteryl esters.58,71,82,103,104,106,172,183– 185 Phospholipid efflux appears to precede cholesterol efflux.106 As a result, apolipoprotein-mediated lipid efflux lipidates apolipoproteins, producing HDL-like lipoproteins with electrophoretic pre-β- and pre-α-mobilities.103– 105,107 Because two-dimensional PAGGE does not readily separate lipid-free apo A-I and lipid-poor pre-β-LpA-I, it is not possible to say whether pre-β-LpA-I functions like lipid-free apolipoproteins, that is, induces cholesterol efflux, or if this particle is itself the primary product of apo A-I-induced lipid efflux.68,70,82 Similarly LpA-IV-1 and γ-LpE appear to be immediate precursors or products of the lipid efflux induced by apo A-IV and apo E, respectively.74,75,77,82
Apolipoprotein-mediated lipid efflux involves nonspecific desorption of plasma membrane lipids, that is, microsolubilization, and specific interactions with plasma membrane proteins.174 It is cell-specific and only takes place in growth-arrested and cholesterol-enriched cells.82,104 Lipid-free apolipoproteins remove phospholipids and cholesterol from normal human skin fibroblasts, macrophages, and smooth-muscle-cell-derived foam cells,58,71,82,103,104,106,172,183– 186 but not from erythrocytes, native smooth muscle cells of various species, and fibroblasts of Tangier disease patients.58,186– 189 Moreover, lipid-free apolipoprotein mediated cholesterol efflux is completely suppressed by low temperature, by partial proteolysis of cell membranes, and by treatment of cells with monensin or brefeldin A which interfere with the regular function of the Golgi apparatus and with intracellular vesicular transport.58,142,148,183 It has been suggested that lipid-free apolipoproteins and pre-β1-LpA-I specifically release cholesterol which is located in caveolae.134,146 Activation of protein kinase C (PKC) enhances and inhibition of PKC suppresses apolipoprotein-mediated cholesterol efflux.186,190– 192 Cyclic AMP (cAMP) was also found to increase apolipoprotein-induced cholesterol efflux from two mouse macrophage cell lines.193– 195 In RAW264 cells this was associated with internalization and resecretion of apo A-I.193 For these reasons and because apo A-I binds to cell membrane proteins and induces the generation of the PKC-activator diacylglycerol, it has been hypothesized that apo A-I binds to a signal-transducing cell-surface receptor, and that this binding facilitates the translocation of cholesterol from intracellular compartments to the plasma membrane.82,172,196– 199 The nature of this receptor is unknown. Because both cholesterol efflux from cells and the ATP binding cassette transporter 1 (ABC1) are defective in Tangier disease, it is likely that ABC1 plays an important role in apolipoprotein-mediated efflux.200– 205 In support of this, apo A-I-mediated cholesterol efflux was severely decreased by inhibition of ABC1 with either antisense oligonucleotides or pharmacologic compounds (4,4-diisothiocyanostilbene-2,2-disulfonic acid or sulfobromophthalein) and increased by overexpression of ABC1.203
Native and reconstituted lipid-rich HDL induce both specific and nonspecific forms of cholesterol efflux. Partial proteolysis of either HDL or cells does not fully prevent HDL-mediated cholesterol efflux, which is slow, unsaturable, and bidirectional, and thus appears to occur by aqueous diffusion.71,82,136,172,173,181 Esterification of the released cholesterol by LCAT prevents the rediffusion of cholesterol from HDL back to the plasma membrane, and thus enhances net cholesterol efflux.206 Expression of SR-BI increases HDL-mediated cholesterol efflux.207 Binding of HDL to SR-BI appears to reorganize the lipid organization in the plasma membrane and to facilitate the bidirectional flux between HDL and plasma membrane.208 The net movement of cholesterol appears to depend on the relative activities of extracellular lipid transfer enzymes (LCAT, PLTP, CETP) and intracellular enzymes which metabolize cholesterol (e.g., ACAT).173
HDL-mediated cholesterol efflux also shares some properties of lipid-free apolipoprotein-mediated cholesterol efflux. In the presence of HDL, intracellularly stored cholesteryl esters are reduced.170,171,194,195 After incubation of lipid-enriched cells with brefeldin A or the PKC inhibitor sphingosine, HDL-mediated cholesterol efflux is reduced by about 50 percent.58,142,148,191 Both native and reconstituted HDL elicit various signal transduction pathways that may activate intracellular lipid transfer processes.186,190– 192,209– 217 HDL induces the hydrolysis of phosphatidylcholine (PC) and phosphatidylinositol (PI) by phospholipases C and D, and thereby the generation of diacylglycerol (DAG, phosphatidic acid (PA), and inositolphosphates (IP). DAG activates PKC, which was shown to stimulate the translocation of newly synthesized cholesterol to the plasma membrane as well as cholesterol efflux.190,192,209,212,214 In macrophages, HDL increases the concentration of cAMP, resulting in activation of protein kinase A, which may activate the hydrolysis of cytosolic cholesteryl esters by NCEH.169,218– 221 It is not known whether these specific effects of HDL on cellular cholesterol metabolism are induced by the intact particle or by apolipoproteins that have dissociated during the incubation with cells.
Another controversial mechanism of HDL-mediated cholesterol efflux is retroendocytosis. After uptake into clathrin-coated endosomes, it is postulated that HDL are directed into a nonlysosomal route and enriched with lipids for final resecretion.193,222,223