Role of the Liver in the Degradation of Lipoproteins

Transcription

1 GASTROENTEROLOGY 1985;88: PROGRESS ARTICLE Role of the Liver in the Degradation of Lipoproteins ALLEN D. COOPER Department of Medicine. Stanford University School of Medicine, Stanford, California Lipids serve both as a major fuel source and as structural components of cell membranes. Not surprisingly then, their homeostasis is carefully maintained by a number of interrelated metabolic pathways. The insolubility of lipids in aqueous fluids adds complexity to the transport process by creating the need for a carrier system. The carrier system of protein-lipid aggregates or lipoproteins has been thoroughly studied for many years. The liver is known to playa central role in the regulation of the synthesis, degradation, and storage of lipids and of lipoproteins. Several diseases including atherosclerosis and cholethiasis can be attributed to disturbances in one or more of these metabolic pathways. Lipoprotein metabolism overall is a broad field and cannot be fully summarized in such a review as this. In Table 1, important terms are defined and abbreviations are listed to aid the reader. Figure 1 gives an overview of some of the pathways involved in the overall regulation of lipoprotein metabolism. Detailed information concerning the origin, metabolism, or structure of lipoproteins can be obtained from one of several authoritative sources (1-4). Lipoproteins are divided into classes according to their buoyant density upon ultracentrifugation. The classes are, in order of increasing density, chylomicrons, very low-density lipoproteins (VLDLs), low-density lipoproteins (LDLs), and high-density lipoproteins (HDLs). The buoyant density is inversely proportional to the triglyceride content of the particle. Thus, chylomicrons are composed largely of triglyceride whereas HDLs have virtually none. These are somewhat arbitrary separations, and it is now appreciated that there is significant structural and functional heterogeneity within the classes. In addition, the Received April 10, Accepted August 8, Address requests for reprints to: Allen D. Cooper, M.D., Department of Medicine, Room S-069, Stanford University Medical Center, Stanford, California The original research cited in this review article was supported by grants AM and HL from the National Institutes of Health and by a grant from the American Heart Association by the American Gastroenterological Association /85/$3.30 system is a dynamic one with one class producing another during its metabolism. Similarly, apoprotein metabolism is not a static process. Thus, the A apoproteins (apo A) are produced by liver and intestine, are secreted on chylomicrons or on HDLs and p o s ~ iit! bunassociated l y form and then transfer to HDLs. There are two forms of apo B; one, a smaller form, is synthesized by intestine and the other, a larger form, by liver. This protein seems to be required for the release of the lipoprotein from a cell and stays with the lipoprotein during its metabolism. The C apoproteins are small peptides that are synthesized by liver and transfer among the lipoprotein classes, controlling their metabolism by stimulating and perhaps inhibiting lipolytic enzymes. The E apoprotein is synthesized by liver, and perhaps other tissues, is secreted on VLDLs and HDLs, and by transferring to triglyceride-rich lipoproteins, plays a key role in directing their metabolism. The role of the liver in the synthesis and secretion of lipoproteins was established in the late 1950s and became the focus of a great deal of research (5-7). It was appreciated that the rate of synthesis and secretion of these particles by liver was an important determinant of their circulating level. It was, however, not until the 1970s that investigators began to examine the regulation of lipoprotein degradation in detail and began to assess the role of the liver in this process. Secretion and degradation are equally important in determining lipoprotein levels because, in the steady state, the rate of synthesis must equal the rate of degradation, and a change in either will cause a change in the circulating level. In liver disease there are profound and characteristic changes in lipoprotein composition and metabolism. For example, there is a loss of HDL in acute parenchymal disease and the appearance of an unusual lipoprotein, LPX, in obstructive disease (see Reference 4 for a discussion.) Understanding how these changes occur and their significance in pathogenesis are important challenges for future research. The purpose of this review is to provide a concise, but up-to-date summary of the role of the liver in the

2 January 1985 HEPATIC LIPOPROTEIN DEGRADATION 193 Table 1. Terms 1. Lipoprotein: macromolecular complex of lipid and protein of fixed composition. 2. Lipoprotein class: a group of lipoproteins separated by physical means, usually density, size, or electrophoretic mobility. It is important to note that within the classes there may be considerable heterogeneity of the particles in terms both of composition and metabolism. The common classes are described below. 3. Chylomicron: large ( A) triglyceride-rich particles of intestinal origin. 4. Very low-density lipoprotein (VLDL): A particles that are rich in triglyceride and are primarily of hepatic origin. 5. Low-density lipoprotein (LDL): 200 A particles that are rich in cholesterol. This is the most abundant class in humans and its level in serum correlates with the prevalence of atherosclerosis. 6. High-density lipoprotein (HDL): small ( A) particles that are rich in cholesterol and protein. They are the most abundant class in many species and their level in serum correlates inversely with the incidence of atherosclerosis. 7. Apoproteins (apo): the protein components of lipoproteins. These have been divided up into groups. A: the A apoproteins are the principal apoproteins of HDL. Apo A-I is most abundant; also present are A-II and A-IV. B: the B apoprotein is present on chylomicrons and VLDLs and is the sole protein constituent of LDLs. Two forms are known; in human, B100 is synthesized by liver and B4B by intestine. C: These are small (mol wt 8,000-10,000) apoproteins. Three different proteins, C-I, C-II, and C-III, are established. They are present on both VLDLs and HDLs. E: this 33,000 mol wt protein is synthesized in liver and is present in VLDLs and HDLs. There is distinct genetic heterogeneity in humans. 8. Lecithin cholesterol acyltransferase (LCAT): a serum enzyme that converts free cholesterol to esterified cholesterol. It is synthesized by the liver. 9. Acyl-coenzyme A cholesterol acyltransferase (ACAT): a microsomal enzyme that converts free cholesterol to cholesterol ester Hydroxy 3-methylglutaryl coenzyme A reductase (HMG CoA reductase): the enzyme that catalyzes the rate-limiting step of cholesterol synthesis. 11. Golgi-endoplasmic reticulum-lysosome (GERL) region. degradation of lipoproteins. This is an area of very active research and much of the available information in textbooks or even reviews rapidly becomes dated. It is hoped that a concise summary of the field will help to generate new ideas. Each of the lipoprotein classes is treated separately with a focus on the role of the liver in the degradation of that class. Low-Density Lipoproteins In many ways the work of Brown and Goldstein (8-11) on the cellular metabolism of LDL initiated the modern era of our understanding of lipoprotein metabolism. Using fibroblasts from normal individuals and patients with familial hypercholesterolemia, these investigators elucidated the "LDL pathway" (Figure 2), which has served as a model for understanding the transport of a large number of macromolecules at the cellular level. The pathway is initiated by the binding of LDLs to a specific cell surface receptor for the apo B component of the lipoprotein. On many cell types this receptor is localized in a specialized region of the cell surface referred to as a coated pit. Low-density lipoprotein receptors are normally located primarily on the coated pit even when no ligand is present. This is in contrast to several other receptors that migrate to the coated pit region only after they bind a ligand. After the binding of LDL to its receptor, the lipoprotein-receptor complex and a portion of the coated pit bud off and form a small vesicle (endosome or receptosome) within the cell. This process is referred to as internalization. Once in the cell the receptor promptly recycles to the cell surface while the LDL particle and endosome migrate toward the Golgiendoplasmic reticulum-lysosome (GERL) region (12). At this stage the vesicles have a distinctive morphology and are referred to as multivesicular body-like structures (12). They then fuse with primary lysosomes to form secondary lysosomes. The lysosomal enzymes degrade the constituents of the LDLs. The apoprotein is degraded to small peptides and amino acids while the cholesterol ester is hydrolyzed to free cholesterol. There are a number of metabolic consequences of this process. First, the free cholesterol liberated reduces the rate of cholesterol synthesis by inhibiting the activity of the enzyme 3-hydroxy 3-methylglutaryl coenzyme A (HMG CoA) reductase. Second, any free cholesterol in excess of that required for immediate metabolic need is converted to cholesterol ester by the microsomal enzyme acyl CoA cholesterol acyl transferase (ACAT) and is stored in this metabolically inactive form. Third, there is a decrease in the number of LDL receptors (down regulation) expressed on the cell surface. All patients with the disease familial hypercholesterolemia have a defect in this pathway. The defects identified to date have included reduced or absent LDL binding because of defective or absent LDL receptors or defective internalization of the bound LDLs. In all instances this results in failure of LDL catabolism by the high-affinity pathway with consequent accumulation of LDLs in the serum. Further details regarding this process are available in the publications of Brown and Goldstein (8-11). After the initial description of this pathway, it was suggested that the liver did not participate to a ::.ignificant extent in LDL catabolism. This was based on the observation that LDL catabolism in the pig was actually accelerated after hepatectomy (13). This

3 194 COOPER GASTROENTEROLOGY Vol. 88, No.1, Part 1 ( t LOL HDl. t J!\YlOMICRON I REMNANTS CHROMICRONS H 1 0 ~ >--! IIHD' INTESTINE \, \. HOL, /... ;'... HOL "",."..._-- Figure 1. An overview of lipoprotein metabolism. The intestine produces chylomicrons and HOLs. The chylomicrons are partially metabolized by peripheral tissues to form remnants, which are removed by the liver. Some HOL is produced in the process of remnant formation as well. The liver secretes VLOLs and HOLs. Very low-density lipoproteins are converted by peripheral tissue to LOL and VLOL remnants. The latter are removed by liver. Low-density lipoproteins are removed by sterol-requiring peripheral tissue, such as adrenal gland and gonads, and by the liver. Some HOL is probably formed during VLOL metabolism. The fate of HOLs is unknown. concept has been substantially revised over the past several years as considerable evidence for participation of the liver in LDL degradation has accumulated. First, it has been shown that, when placed in primary culture, hepatocytes can express LDL receptors that function similarly to those on the fibroblast (14-16). Second, it was shown that LDL catabolism by perfused liver could be stimulated by treating rats with ethinyl estradiol (17). Moreover, binding studies with membranes prepared from the livers of these. NUCLEUSONDOPLASMIC RETICULUM?. 1) ~ HMG CoA Reductase 2) t Cholesterol Esterification 3) ~ LOL Receptors 4) Ne change in remnant receptor LOL RECEPTOR (apo B,E) ENDOSOME '7"_... r-- LYSOSOME SECONARY ~ ~ O P R I M A R Y MVLB ~ LYSOSOME Figure 2. Hepatocellular lipoprotein metabolism. Low-density lipoproteins bind to specific receptors located on "coated" pits on the cell surface. They are internalized via an endosome and merge to form multivesicularlike bodies. These bodies merge with lysosomes wherein their contents are degraded and the products released. Several metabolic effects ensue. Cholesterol synthesis is suppressed (HMG CoA reductase), cholesterol esterification is stimulated (ACAT), and the number of LOL receptors expressed decreases. There is a distinct pathway for remnant metabolism. This may share many of the same features but the receptor is distinct and its expression is not regulated by the cholesterol content of the cell. After Brown and Goldstein, with permission from the Annual Review of Biochemistry, Vol. 46, 1977 by Annual Reviews Inc.

4 January 1985 HEPATIC LIPOPROTEIN DEGRADATION 195 rats demonstrated the presence of a receptor with similar characteristics to the LDL receptor of human fibroblasts (18,19). The role of the liver in LDL catabolism under normal circumstances was documented by Pittman et al. (20), who developed a technique for quantifying cumulative lipoprotein removal by a tissue in vivo. They conjugated [ 14 C]sucrose to LDL and demonstrated that the LDL conjugate behaved the same as normal LDL. However, the sucrose moiety cannot be degraded by lysosomal enzymes and thus it accumulates in the lysosomes of the tissue that removes the lipoprotein particle. This allows an assessment of the total amount of an injected lipoprotein that was removed by a tissue. By using this method it was estimated that the liver was responsible for -40% of LDL removal. No other organ removed >9% of the LDLs. For each gram of tissue, only the adrenal gland and gonads, organs known to be extremely rich in LDL receptors, accumulated more LDLs. Recent studies with liver cell membranes from dogs, monkeys, and humans (21,22) generally support the concept that there is an LDL receptor on liver membranes although the characteristics of the binding to liver cell membranes have not been entirely similar to the classical 11::)L receptor. Unpublished evidence from our laboratory has documented that there is a typical LDL receptor on human liver membranes. The quantitative assay of this receptor however is confounded by the presence of a loweraffinity, saturable, non-ethylenediaminetetraacetic acid-sensitive LDL binding that is of unknown physiologic significance (23). By using membrane preparations from dog liver, regulation of the hepatic LDL receptor in response to serum cholesterol level and enterohepatic circulation has been demonstrated. It has also been suggested that the number of receptors on liver decreases with age (21). The postbinding events in hepatic LDL metabolism have been studied to a limited extent, primarily using the ethinyl estradiol-treated rat as a model. Chao et al. (24) examined the hepatic metabolism of 125I-labeled LDL by electron microscopy, whereas Handley et al. (25) followed the fate of LDL conjugated with gold. In both studies lipoprotein was initially seen on the plasma membrane microvilli in regions containing coated vesicles. Later the particles were concentrated in multivesicular bodies in the GERL region. Subsequent changes were consistent with lysosomal degradation of the particles. Thus the pathway for LDL degradation in liver seems to be similar to that described in fibroblasts. There is another aspect of LDL catabolism in which the liver may also playa substantial role. It was appreciated almost immediately after the dis- covery of the LDL receptor that all LDL catabolism could not be accounted for by the high-affinity pathway. Clinical evidence for this concept comes from the fact that patients who completely lack LDL receptors ultimately reach a constant LDL level. Since they have a normal or increased rate of LDL synthesis, they must be catabolizing LDL by some non-ldl receptor-dependent pathway. Evidence for the existence of an alternate or low-affinity pathway and partial quantification of it have been provided by studies using modified LDLs. It was found that modification of LDLs by methylation or cyclohexanedione conjugation of the lysine or arginine molecules on the apo B of LDLs prevents the binding of LDLs to the receptor (26). Because the modification can be reversed and binding activity restored, the lack of binding is not due to denaturation of the particle. Thus the clearance of these modified LDL particles after their injection into intact animals should occur by the non-ldl receptor-mediated pathway. Calculation of amounts cleared by the receptor-mediated and receptor-independent pathways can be made by comparing the clearance of native LDLs (both pathways) and modified LDLs (non-receptor-mediated). This approach assumes that the modification does not alter non-receptormediated clearance, which may not always be correct. Nonetheless, by using a variety of different modifications, it has been estimated that under normal circumstances perhaps o n ~ - tof h LDL i r d removal is by a non-high-affinity pathway(s) (27,28). Moreover, after the injection of labeled modified LDLs into animals, the liver is the organ that accumulates the most radioactivity (28). Further evidence that the liver plays a major role in LDL degradation by non-high-affinity receptor pathways comes from studies in the Watanabe hereditary hyperlipemic rabbit. These inbred rabbits are massively hypercholesterolemic and develop a clinical syndrome very similar to that of patients with familial hypercholesterolemia (29). Biochemical studies have revealed that they lack LDL receptors. However, when [14C]sucrose LDL is injected into these animals, its tissue distribution is very similar to that of normal rabbits (30). In these animals the liver is still the main site of deposition of [14C]sucrose suggesting that even in the absence of LDL receptors this organ is the major site of LDL catabolism. In summary, the following conclusions regarding hepatic LDL metabolism seem warranted. First, the liver is quantitatively the major site of LDL catabolism. Second, the liver possesses and can express high-affinity LDL receptors in numbers regulated by metabolic conditions. Third, the liver also plays a major role in non-high-affinity LDL receptor-mediat-

5 196 COOPER GASTROENTEROLOGY Vol. 88, No.1, Part 1 ed catabolism of LDL. However, it is not yet clear how much receptor-mediated, as compared with receptor-independent, LDL catabolism occurs in liver in the normal or in pathological states. Moreover, future research will need to be directed at providing information about the molecular events of this latter pathway and whether it can be regulated. Last, it is not yet known whether there are similar or differi'lnt metabolic consequences of LDL removal by the different pathways (31). Chylomicrons Chylomicrons are large lipoproteins that are formed in intestinal epithelial cells and secreted into the mesenteric lymph through which they ultimately reach the systemic circulation. An overall scheme for their metabolism is given in Figure 3. It is now thoroughly i'lstablished that metabolism proceeds in two stages. Gould et al. (32) demonstrated that dietary cholesterol had a prompt effect upon cholesterol metabolism in liver but not in other tissues. Goodman (33) found that cholesterol from chylomicrons appeared rapidly and virtually quantitatively in the liver. However, studies by Bergman et al. (34) demonstrated that the fatty acids from dietary triglyceride were removed by peripheral tissues and not by the liver. Moreover, several expi'lriments that used perfused livers failed to demonstrate uptake of chylomicrons by isolated liver. In 1970 this paradox was resolved by Redgrave (35) who used eviscerated (functionally hepatectomized) rats to demonstrate the conversion of chylomicrons to a new lipoprotein species. This particle; which he called a chylomicron remnant, was smaller in size than a chylomicron and was markedly depleted of triglyceride, although it retained most of the cholesterol of the precursor chylomicron. Conversion of chylomicrons to remnants is catalyzed by the enzyme lipoprotein lipase, which is present on the surface of capillary endothelial cells in a variety of tissues including adipose tissue and smooth and cardiac muscle. This enzyme is activated by apo C-II and hydrolyzes the triglyceride from the core of lipoproteins to free fatty acids and glycerol (36). The free fatty acids diffuse into the adjacent tissue such as adipose or muscle and are stored or used for energy by the cell. Depletion of the core of the particle leaves excess lipid on the surface. The excess surface phospholipid and cholesterol are believed to bud off and form an HDL particle (37,38). The smaller triglyceride-depleted particle reenters the systemic circulation. Subsequent work has also demonstrated that there is redistribution of apoproteins during the conversion of the chylomicron to a chylomicron remnant (39). The apo B is synthesized in a specific form by the intestine (40,41) and remains with the particle during its conversion. In contrast, apo A-I is also synthesized by intestine (42) but is lost from the chylomicron after it reaches the circulation. Apolipoprotein E and the C apoproteins are synthesized by liver and are acquired by chylomicrons in the capillaries of the intestine (43). During conversion to INTESTINE r -.,., ~... ~ FREE CHOLESTEROL TRIGLYCERIDE... CHOLESTEROL ESTER LYMPH LIVER HMGCoA REDUCTASE HYDROLYSIS... ~... t INHIBITION... ~ ~... CHOLESTEROL ~... ~... ~ CHYLOMICRON O~ REMNANtS ~. CAPILLAR LIPOPROTEIN LIPASE ENDOTHELIAL CELL Figure 3. Dietary lipoprotein metabolism. The bulk of dietary lipids are incorporated into large lipoproteins called chylomicrons. They are secreted into the lymph and then reach the circulation. Under the influence of endothelial lipoprotein lipase, these chylomicrons are depleted of triglyceride and converted into chylomicron remnants. The remnants are rapidly removed and degraded by the liver where their constituents have a number of metabolic effects.

6 January 1985 HEPATIC LIPOPROTEIN DEGRADATION 197 remnants, most of the C apoproteins are lost but the E apoprotein remains with the remnant (see Figure 4). Because apo C-II is required to activate lipoprotein lipase, the acquisition and subsequent loss of apo C-II during the conversion of a chylomicron to the remnant may be an important determinant of the amount of hydrolysis that a chylomicron undergoes. Factors that govern apo C acquisition and loss are poorly understood at this time. Once a remnant is formed it is rapidly removed by the liver. Studies from several laboratories including ours have shown that isolated livers rapidly remove chylomicron remnants under conditions where precursor chylomicrons are not taken up (44-46). The particle is removed as a unit, with simultaneous disappearance of cholesterol ester, free cholesterol, phospholipid, and residual triglyceride (44). There is also rapid removal of the apoprotein constituents, and there does not seem to be preferential uptake of one apoprotein as compared with any of the others (47). The precise nature of the removal process however remains somewhat controversial. A variety of hypotheses regarding the removal mechanism have been proposed. The first suggests that the process is in many ways similar to LDL transport and involves binding to a cell surface receptor followed by internalization and degradation. In support of this, it has been shown that the transport of chylomicron remnants by perfused livers exhibits kinetics compatible with a saturable, energy-dependent process (48,49). Unlike the LDL pathway, however, remnant removal is not regulated by the cholesterol content of the cell (48-50). The fact that remnant removal is normal in the Watanabe hyperlipemic rabbit, which lacks LDL receptors, is further strong evidence for the existence of a remnant removal mechanism distinct from the LDL receptor pathway (51). The existence of a cell surface receptor relatively specific for a chylomicron remnant has been suggested by studies from our laboratory in which saturable and specific binding of chylomicron remnants to liver plasma membranes was found (52). The nature of the recognition site on the particle is not entirely resolved. Work by Mahley and coworkers (21,22) that used liver membranes from dogs, monkeys, pigs, and humans also suggests that there is a receptor distinct from the LDL receptor (21,22). They suggest that this receptor recognizes only apo E (E receptor) in contrast to the LDL receptor, which can recognize both the Band E apoproteins (B,E receptor) (53). Studies from our laboratory also suggest that apo E has a role in the binding of a remnant to the plasma membrane (54). Experiments in perfused liver conducted by Windler et al. (55) suggest that it is the ratio of E to C apoproteins that best predicts the ability of a particle to be removed by perfused liver, whereas work by Figure 4. Apoprotein changes during remnant metabolism. When synthesized by intestine, chylomicrons contain primar i1y apo B and the A apoproteins (I, II, IV). Upon contact with the circulation they lose the A apoproteins and acquire apo E and the C apoproteins from HDLs. After lipolysis they lose the C apoproteins. Their removal by liver appears to require apo E. Quarfordt and colleagues (56,57) that used triglyceride emulsions also suggests an important role for C apoprotein in inhibiting the removal of these emulsions by the liver regardless of whether the E apoprotein is present or not. An important role for apo E in the metabolism of triglyceride-rich lipoproteins is provided by a series of elegant genetic and structural studies. It has been known for some time that apo E is heterogeneous when examined by polyacrylamide gel electrophoresis and isoelectric focusing. Zannis and Breslow (58) demonstrated that there are three distinct alleles, Ez, E 3, and E 4. Thus, an individual can have one of six different genotypes (E-2, E-2 ; E-3, E-3; E-4, E-4; E2, E- 3; E-2, E-4; E-3, E-4). Patients with dysbetahyperlipidemia (type III hyperlipemia) are of the homozygous (E-2) genotype. Weisgraber and colleagues (59) sequenced apo E from patients with the various homozygous genotypes and demonstrated that the variability was due to cysteine-arginine substitutions. Metabolic studies have shown that there is decreased binding of apo E to LDL receptors (60) and possibly to remnant receptors (61). This could account, at least in part, for the hyperlipemia in these patients. In evaluating all of these experiments, certain considerations are necessary. First, distinguishing the remnant pathway from the LDL pathway and ensuring that there is no LDL receptor activity present when studying this pathway has proven a difficult task because apo E has a very high affinity for the LDL receptor (about 20-fold higher than apo B) (62). Additionally, when modified or artificial lipoproteins are used, nonphysiologic pathways may become responsible for transport of the lipid and possibly the apoprotein.

7 198 COOPER GASTROENTEROLOGY Vol. 88, No.1, Part 1 A second formulation would suggest an important role for the hepatic lipase in accomplishing lipoprotein removal. Work from Borensztajn's laboratory (63,64) has suggested that alteration of the phospholipid on the particle, possibly mediated by the hepatic lipase, may play a critical role in rendering a lipoprotein suitable for removal by this pathway. The enzyme monoglycerol acyltransferase has also been suggested to have a role in this process (65). Moreover, patients with familial hepatic lipase deficiency accumulate intermediate-density lipoproteins that could be remnants (66). However, such a pathway would not explain how the apoproteins are removed and thus it might be necessary to postulate a two-step mechanism in which there is extensive hydrolysis of the particle by a lipase and then a second step in which the residual coat material including the apoproteins is removed. A third, somewhat different, postulate has been put forth by Sparks and Marsh (67) who have suggested a two-step process, in which the apo E serves as a ligand for binding that leads to removal of only the apo B and the lipid with release (or possibly resecretion) of a new particle containing the apo E and some lipid. Evidence for this pathway comes from studies in which disappearance of the various apoproteins was followed after incubation of VLDLs with postheparin plasma to create remnants. Rapid disappearance of the smaller apo B (which arises in intestine) was observed, with much slower disappearance of the large apo B (which comes from liver) and partial disappearance of the E apoprotein. However, in these experiments it remained possible that there was some redistribution of the apo E before remnant formation. The precise contribution of the different hepatic cell types to remnant removal is not completely resolved. Several studies have demonstrated the participation of the hepatocyte in the process (68,69). Other studies suggest a role for Kupffer cells in the process (70). Unpublished quantitative studies in our laboratory have suggested that no more than 30% of remnants are removed by nonparenchymal cells. Because peripheral blood macrophages have receptors for a variety of chemically and physiologically modified lipoproteins, the participation of this cell type in remnant metabolism raises interesting speculations about the possible role of dietary particles in atherogenesis (71-73). Most studies, however, have used unscreened chylomicrons and hence it is possible that denaturation or agglutination of the particles occurred before their injection into the experimental animal and this led to their recognition by macrophages. Thus the entire question of the physiologic role of macrophage pathways in chylomicron metabolism should provide fertile ground for future research. The final quantitative solution to this important question awaits using carefully screened lipoproteins or other novel techniques. Once the lipoprotein particle has been removed by the liver, its fate has been partially elucidated. Early studies by Stein et al. (74), in which [14C]cholesterol ester containing chylomicrons was injected into rats followed by autoradiography of liver sections, demonstrated radioactive material not only at the plasma membrane but also within the parenchymal cell primarily associated with lipid droplets. Again these observations are compatible with a receptor-mediated endocytic process although these investigators did not note concentration in lysosomes. Our studies (48) revealed that the hydrolysis rate of removed remnant cholesterol ester was ~ 0. of 5 % the removed mass per minute. This rate was linear over the concentration of remnants studied and appeared to be far below the possible degradation rate when the kinetics of all of the available cholesterol esterase within the liver was taken into consideration. Thus it was suggested that movement from the cell surface to a lysosomal compartment might be a rate-determining step, at least in the initial disposition of newly removed dietary cholesterol. Evidence for a role of the lysosome in this process has been provided by studies by Floren et al. (75), who found that chloroquine slowed the degradation of chylomicron components by liver. Together, these data are consistent with intracellular processing similar to that of LDL. However, the data are far from conclusive and this process certainly warrants further study. An interesting abnormality of remnant metabolism may occur with malignant degeneration. It was established by Siperstein (76) that hepatomas lack the normal feedback inhibition of cholesterol synthesis induced by dietary cholesterol. Harry et al. (77) suggested that this might be due to a failure of dietary cholesterol to reach the malignant liver. The intracellular steps in the regulation of cholesterol synthesis have been found to be intact in hepatomas (78). Our laboratory has recently shown that, although hepatomas have receptors that recognize and transport chylomicron remnants, the number of these receptors is markedly reduced compared with that of normal liver (79). This quantitative loss of receptors can account for the apparent loss of feedback inhibition. Very Low-Density Lipoproteins Very low-density lipoproteins are secreted primarily by the liver (7) and thus one might anticipate that they do not return to the liver for catabolism. However, this may not be entirely correct

8 January 1985 HEP A TIC LIPOPROTEIN DEGRADATION 199 (Figure 5). There appear to be three relatively distinct pathways of VLDL metabolism. Although all species may utilize each of the pathways, there are major differences as to which pathway predominates in a particular species. A portion of VLDL appears to be metabolized by a pathway that is analogous to the chylomicron remnant pathway, with removal by the liver, after hydrolysis of triglyceride by lipoprotein lipase in peripheral tissue (80,81). In the rat, this is actually the primary pathway of VLDL metabolism and as much as 90% of VLDL may be metabolized via this route (80). In this species there is evidence that it is the chylomicron remnant receptor that removes VLDL remnants in that the two particles compete for the same membrane binding site and can reduce each other's uptake by the perfused rat liver (81). In the Watanabe rabbit, which lacks an LDL receptor, VLDL clearance is delayed whereas chylomicron clearance is not affected, suggesting that in the rabbit, once a VLDL remnant is formed, it is not a ligand for the chylomicron remnant receptor, but that in this species VLDL remnants are normally cleared by the LDL receptor (82). The third mechanism of VLDL metabolism, which predominates in humans, is conversion to LDL (83,84). In humans between 50% and 90% of VLDL is converted to LDL. This conversion seems to involve not only peripheral tissue lipoprotein lipase but also the hepatic lipase. This latter enzyme does not require apo C-II for activation and thus a VLDL, which has lost its apo C-II under the influence of lipoprotein lipase, can lose its residual triglyceride under the influence of hepatic lipase and become an LDL particle (85). Patients lacking the hepatic lipase accumulate triglyceride-rich LDLs as well as particles that are intermediate in size between VLDLs and LDLs, often referred to as intermediate-density lipoproteins (IDL) (66). The factors that control the relative amounts of VLDLs catabolized by the various pathways are unknown. One good candidate for a role in this process is apo B. The intestine secretes only a lower-molecular-weight form of apo B (B L, B 48 ) (40,86). The liver in humans secretes only a higher-molecular-weight form of apo B (B H, B ldo ) (40), but in the rat the liver secretes large and small apo B (87). Thus the presence of small apo B may be involved in directing particles down the remnant pathway and to the liver (88). In addition, the amount of apo E retained on the particle as it undergoes lipolysis may also be critical. Thus rat VLDL remnants retain apo E during formation and remain as ligands for the remnant receptor whereas rabbit and human VLDLs may lose their apo E and thus can be ligands for only the LDL receptor. There is, however, no experimental evidence for this conjecture. High-Density Lipoprotein Interest in HDL metabolism was stimulated by the observation that the serum HDL level is a negative risk factor for cardiovascular disease (89,90). This was consistent with the role for HDL proposed LIVER Tissues with ldl receptor lipoprotein lipase CAPILLARY ENDOTHELIAL CELL Figure 5. Very low-density lipoprotein metabolism: VLDLs are secreted by liver and undergo lipolysis on endothelial tissue. Their remnants may be removed by liver (primarily in the rat) or further modified to form LDLs (primarily in humans). When secreted by liver, VLDLs contain apo B and some apo E and apo C. The particles seem to lose apo E to HDLs and acquire apo C from HDLs when they reach the circulation. During lipolysis they lose C apoproteins and, in humans, during conversion to LDLs lose apo E as well.

9 200 COOPER GASTROENTEROLOGY Vol. 88, No.1, Part 1 by Glomset (91) in a process termed reverse cholesterol transport. In this formulation excess cholesterol in peripheral tissue is incorporated into the cholesterol ester of HDL by the enzyme LCAT (91). The HDL is then removed by the liver and its cholesterol is excreted or catabolized to bile salts. To date direct evidence for this hypothesis is lacking and HDL metabolism has proven more complex than anticipated. This is due in part to the fact that the lipoproteins isolated in the HDL density range are quite heterogeneous both in terms of composition and metabolism (see Figure 6). High-density lipoproteins are thought to arise from at least four distinct sources: direct secretion from liver (92) and intestine (93) and from the excess surface of chylomicrons and VLDLs, created when their cores are depleted of triglyceride by lipolysis (37,38). Moreover, the components of the HDLs can be transferred back and forth between the other lipoprotein classes making attempts to follow the fate of an HDL particle as a unit difficult. Thus it is not clear whether HDL particles are metabolized as units or whether their constituents have separate metabolic fates. Evidence in support of the latter possibility has been accumulating for the past several years. First, it was established that the A apoproteins have a longer half-life than the C apoproteins (3). Second, free cholesterol from HDLs disappears from plasma and appears in bile more rapidly than cholesterol ester from HDLs (94). Third, by using a labeling method similar to the [14C]sucrose LDL technique, it has recently been shown that more apo A-I from HDL is removed by kidney than is removed by liver, whereas HDL cholesterol ester (95) appears principally in liver. Despite the unsettled state of the art, there is reason to believe that the liver plays a role in HDL catabolism. Sigurdsson et al. (96) demonstrated that 125I-labeled HDLs were removed and catabolized by isolated perfused liver. They noted that the rate of removal was far too low to account for the bulk of HDL turnover in vivo. Evidence for removal of HDL cholesterol by extrahepatic sterol using tissue such as adrenal and gonad in vivo (97) and in vitro (98,99) has been provided by several laboratories. The nature of this transport process is incompletely understood, but some evidence for the participation of apo A-I and an apo A-I receptor has been provided (98,100). Preliminary publications suggest that a similar process occurs in liver (101). It is also established that apo E-rich HDL (HDLc) is rapidly removed by liver (53). It is likely that both the LDL receptor mechanism and the chylomicron remnant transport mechanism can mediate this latter process (21). A second type of HDL-hepatocyte interaction has been described in a thorough study of HDL binding by isolated porcine hepatocytes (102). It was found that there was high-affinity HDL binding but that this INTESTINE VLDL REMNANT o : : : : : : : = = : : : : : : : CHYLOMICRON ; : : : : : : : : ~ ~ o \..0 CAflLLARY CHYLOMICRON ENOOTHELIAL REMNANT CELL Figure 6. High-density lipoprotein metabolism: HDLs are formed from four distinct sources. There is direct secretion by liver and intestine as well as formation via the catabolism of chylomicrons and VLDL. High-density lipoproteins contain apo A-I and apo A-II as their primary protein. They also contain variable amounts of apo E and C apoproteins. High-density lipoproteins are heterogeneous in terms of composition and possibly metabolism. Several subclasses including HDL2 and HDL3 are now recognized.

10 January 1985 HEPATIC LIPOPROTEIN DEGRADATION 201 led to substantially less degradation of HDL than did a comparable amount of LDL binding. Biesbroeck and colleagues (103) have also suggested, based on studies with nonhepatic tissue in culture, that there is an HDL receptor that does not lead to internalization and degradation. Such a process could lead to transfer of lipid without endocytosis of the particle. Another mechanism whereby HDL constituents could reach the liver is through the remnant and LDL pathways. Because HDL apoproteins transfer back and forth between HDLs and triglyceride-rich lipoproteins, any apoprotein that remains with the remnant particle will be catabolized after remnant removal. Similarly, it is well established that cholesterol ester is transferred from HDL to lowerdensity lipoproteins after its formation from free cholesterol of HDL. This reaction is catalyzed by the enzyme LCA T and one or more transfer proteins (91, ) (Figure 7). Thus there are a number of established and potential mechanisms whereby HDL interacts with the liver and some, if not all, of its constituents become catabolized there. However, the quantitative and physiologic significance of these various mechanisms remains to be established. The available quantitative information regarding HDL metabolism by liver suggests that the liver may not be the principal site of HDL apoprotein degradation. Glass et al. (95) coupled a nonmetabolizable marker [ 125 1]cellubiose to apo A-I, reconstituted the apo A-I with HDL, and followed the accumulation of in tissue, much as was done previously with [ 14 C]sucrose LDL. They made the surprising observation that the kidney was the principal site of A-I removal (35%) and that liver was second with 25% of the counts. On a per gram of tissue basis, liver was less active not only than kidney but was also less active than ovary, adrenal, and spleen. On the other hand the same authors noted that cholesterol ether from HDL, a nonmetabolizable marker of the core of the particle, did not accumulate in kidney, but was most abundant in liver. In summary, little is settled concerning HDL catabolism but it seems likely that once the various proposed mechanisms are evaluated and understood the liver will be found to playa significant role in this process and may be the ultimate site for disposition of much of the cholesterol ester carried by these particles. Other Hepatic Processes That Affect Lipoprotein Degradation In addition to directly removing and catabolizing lipoproteins, the liver contributes to lipoprotein catabolism in several other ways. First, by synthesizing and secreting apoproteins, it creates the signals that ultimately direct lipoprotein catabolism. Apoprotein B, apo E, apo A-I, and the C apoproteins that are synthesized in the liver have been shown, or suggested, to direct the catabolism of various lipoprotein fractions. Thus processes that regulate their synthesis will ultimately affect lipoprotein degradation. The enzyme LCAT, which is synthesized and secreted by liver, is responsible for esterifying free cholesterol in the serum (91) and in conjunction with transfer proteins, which may also be synthesized in liver, is believed to playa role in moving cholesterol from peripheral tissue to lipoproteins and ultimately to liver (see Figure 7). Last, hepatic lipase may have an important role in the formation of LDL from VLDL and may also have a role in remnant metabolism. Thus, hepatic diseases that affect these proteins lead to profound disturbances in lipoprotein metabolism which may largely be the result of disturbed catabolism. HDL DISC 1 ~ CELL MEMBRANE exchange of free cholesterol allows vacancy to be f illed + LCAT - ~ ~ ~ _ Tronsfer Cholesterol ~ C h o l e s t ecomplex r o ""'--""""'-ester l core t ester In core moves HDL cholesterol Sphere ester (vacant site Chylomicron. for cholesterol VLDL or LDL onsurfoce) / LDL to Peripheral Tissue Remnant to Liver Figure 7. Reverse cholesterol transport. Free cholesterol from a cell enters the phospholipid monolayer of HDLs. It is esterified by the enzyme LCAT and then moves to the core of the HDLs. From there it may be transferred to a lower-density lipoprotein class and thus to the liver. From Cooper (4). Reproduced with permission of the publisher.

11 202 COOPER GASTROENTEROLOGY Vol. 88, No. 1, Part 1 Importance of Lipoprotein Catabolism in Hepatic Lipid Metabolism Given the many uncertainities in the area at present, it is difficult to provide firm quantitative estimates for the amount of lipoprotein-derived lipid processed by the liver. However, even rough estimates based on what is currently believed suggest that a large amount of lipid enters the liver by this route and must be metabolized. For example, in an average diet, 90% of the 500 mg of dietary cholesterol (400 mg) and 20% of the 80 g (16 g) of dietary triglyceride directly reach the liver via the remnant pathway each day. Low-density lipoprotein cholesterol catabolism is ~ 2 g - per 3 day and over half of this occurs in the liver. How do fluctuations in this input affect hepatic metabolism? This important problem has not been addressed in depth. It does appear that, although the liver can serve, at least to a degree, as a buffer to protect the rest of the body from wide fluctuations in lipid levels, this capacity can be overcome and alterations in whole body metabolism induced. For example, cholesterol feeding in some species leads to prompt changes in biliary lipid composition. Moreover, we have found that the ratio of triglyceride to cholesterol in remnants rather than the absolute amount of either alone can be an important determinant of hepatic lipoprotein production (107) and rate of cholesterol synthesis. Quarfordt and colleagues (108) have found that fatty liver may be induced rapidly by increasing the amount of triglyceride reaching the liver and have proposed that there may be a "prelysosomal" fatty liver in addition to the other metabolic causes of fatty liver. Taken together then it appears that the liver is the major organ of lipoprotein catabolism. 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Increased binding of low density lipoprotein to liver membranes for rats treated with 17 a-ethinyl estradiol.) BioI Chern 1979;253: Pittman RC, Attie AD, Carew TE, Steinberg D. Tissue sites of degradation of low density lipoproteins: application of a method for determining the fate of plasma proteins. Proc Natl Acad Sci USA 1979;76: Hui DY, Innerarity TL, Mahley RW. Lipoprotein binding to canine membranes. Metabolically distinct apo-e and apo B,E receptors. I Bioi Chern 1981;256: Mahley RW, Hui DY, Innerarity T1. Two independent lipoprotein receptors on hepatic membranes of dog, swine and man. I Clin Invest 1981;68: Erickson SK, Kane IP, Hardman D, Blum C, Cooper AD. Lipoprotein binding to human liver cell membranes. Clin Res 1982;30: Chao YS, lones AL, Hradek GT, Havel RJ. Autoradiographic localization of the sites of uptake, cellular transport, and catabolism of low density lipoproteins in the liver of normal and estrogen-treated rats. 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