SEMINARS IN LIVER DISEASE-VOL.
12, NO. 4, 1992
Hepatic Clearance of Plasma Low Density Lipoproteins
Complications of atherosclerosis, most notably coronary heart disease and myocardial infarction, are a major cause of morbidity and mortality in Western populations. Of the many risk factors that have been associated with the development of atherosclerosis and coronary heart disease, an elevated concentration of low density lipoproteins (LDLs) in the plasma appears The concentration of LDL to be one of the most irn~0rtant.l.~ in plasma is determined by the rate at which LDL enters the plasma relative to the rate at which LDL is cleared from plasma by the various organs of the body. LDLs circulating in the plasma are formed during the metabolism of triglyceride-rich very low density lipoprotein^^,^ (VLDL), which in turn are secreted by the liver (Fig. 1). The primary function of VLDL is to transport triglyceride from the liver to peripheral sites of utilization and storage. Most of the triglyceride in the VLDL particle is hydrolyzed by lipoprotein lipase, an enzyme located on the surface of endothelial cells in skeletal muscle, heart, and adipose tissue. VLDL are thereby converted to VLDL remnants, a portion of which is rapidly cleared by LDL receptors in the liver, while the remainder are metabolized to LDL, apparently as a result of the continued action of lipoprotein lipase and hepatic triglyceride lipase. In most animal species, the majority of VLDL is rapidly cleared from plasma as VLDL remnants, and plasma LDL concentrations are low.' In humans, however, 80 to 90% of VLDL is ultimately metabolized to LDL particles, which have a much slower t ~ r n o v e r . ~ LDL is taken up into tissues by at least two distinct transport processes. One of these is receptor dependent and involves the interaction of apolipoprotein (apo) BlOO of LDL with specific receptors on the cell surface followed by clustering of these receptors in coated pits and internalization via an endosoma1 In the fibroblast, the LDL receptor is synthesized on membrane-bound ribosomes, glycosylated in the Golgi apparatus, and then inserted into the plasma membrane. Within 10 minutes, LDL receptors gather into coated pits that invaginate to form endocytic vesicle^.^ Multiple endocytic vesicles fuse to form larger endosomes where the LDL particle dissociates from its receptor as a result of a fall in the pH within the endosome. The LDL receptor, along with a number of other receptors, clusters in a portion of the endosomal membrane that pinches off and returns the receptors to the cell surface while the LDL particles are delivered to lysosomes where they are hydrolyzed to amino acids, fatty acids, and cholesterol. The liberated cholesterol is used to support membrane biosynthesis and also serves as a precursor for bile acids and steroid hormones in the liver and endocrine organs, respectively.
From the Department o f Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas.
Reprint requests: Dr. Spady, Department of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235-8887.
A second process, termed "receptor-independent transport," apparently does not involve specific receptors. In contrast to receptor-dependent LDL uptake, receptor-independent transport is nonsaturable and appears not to be regulated. Rates of receptor-independent LDL uptake are generally low; however, since receptor-independent transport increases as a linear function of plasma LDL concentrations, uptake via this pathway can predominate at high plasma LDL levels. Although the LDL receptor pathway was first described in cultured fibroblasts, it is now known that the majority of LDL receptors are located in the liver and that the liver is the major site of LDL catabolism. Thus, the liver plays a central role in controlling plasma LDL concentrations in that it is the source of LDL via VLDL, is involved in the conversion of VLDL to LDL, and is the major site of LDL catabolism. This review will summarize current information regarding the role of the liver in the catabolism of LDL with particular emphasis on the mechanisms whereby dietary and genetic factors interact to control the rate of receptor-dependent LDL uptake by the liver.
CONTRIBUTION OF THE LIVER TO TOTAL BODY LOW DENSITY LIPOPROTEIN CATABOLISM With the development of radiolabeled markers that are retained by tissues after uptake,& it has become possible to measure rates of LDL uptake accurately in all of the major organs of the body in vivo." Total LDL uptake by individual tissues or the whole body can be determined using homologous LDL, and the receptor-independent component can be determined using LDL that has been modified to eliminate completely its interaction with the LDL receptor. This approach is based on the observation that methylation or glucosylation of the lysine residues of apoB 100 completely eliminates binding ~ , ' difference ~ between total and reto the LDL r e c e p t ~ r . ~The ceptor-independent LDL uptake represents the receptor-dependent component of total LDL uptake. Rates of receptor-dependent and independent LDL uptake by individual organs and the whole body have now been determined in several species, including the hamster, rat, mouse, rabbit, dog, and cynomolgus monkey. A similar tissue distribution of LDL uptake has been observed in all of the species studied thus far",14.1s(Spady D K and Dietschy J M: Unpublished observations). When expressed per gram of tissue, rates of total LDL uptake are highest in the liver, small intestine, endocrine organs, and spleen." Rates of LDL uptake in most other tissues are quite low; indeed, LDL uptake is virtually undetectable in the major tissue compartments of the body such as fat, skeletal muscle, skin, and brain. When whole organ weights are taken into account, the liver is clearly the most important site of LDL catabolism, accounting for approxi-
Copyright O 1992 by Thieme Medical Publishers, Inc., 381 Park Avenue South, New York, NY 10016. All rights reserved.
373
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DAVID K. SPADY, M.D.
12, NUMBER 4, 1992
Extrahepatic tissues FIG. 1. Processes involved in the production and catabolism of LDL and their relationship to the major pathways of net sterol input and loss from the liver. Dietary cholesterol and triglycerides enter the body and are transported within chylomicron (CM) particles to peripheral tissues where, after hydrolysis of most of their triglyceride, the particles are converted to chylomicron remnants that are rapidly removed from plasma, primarily by the liver. Similarly, the liver exports cholesterol and triglyceride within VLDL particles. Following loss of triglyceride in peripheral tissues, a portion of VLDL is converted to VLDL remnants, which are rapidly cleared by the liver, while the remainder of VLDL is further metabolized to LDL particles, which are also cleared primarily by the liver. Finally, secretion of cholesterol into bile, either directly or after conversion to bile acids, provides the only significant route for cholesterol elimination from the body. 7~-OHase:7~hydroxylase.
FIG. 2. Rates of receptor-dependent and -independent LDL uptake by individual organs and the whole body. Rates of LDL uptake were measured in hamsters using primed infusions of [125Utyramine cellobiose-labeled homologous LDL (total LDL uptake by receptor-dependent and receptor-independent pathways) and methylated human LDL (the receptor-independent component of total LDL uptake). Rates of LDL uptake are expressed as the micrograms of LDL cholesterol taken up per whole organ per hour per 100 gm body weight. These values were obtained in animals maintained on standard low-cholesterol, low-triglyceride rodent diet. SB: small bowel.
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SEMINARS IN LIVER DISEASE-VOLUME
mately three fourths of total LDL turnover, as illustrated in Figure 2 for data obtained in the hamster. Moreover, the high rate of LDL uptake by the liver is largely receptor dependent. Thus, in animals fed a low cholesterol diet 80 to 90% of total body receptor-dependent LDL transport takes place in the liver. A technical problem that may lead to underestimation of the importance of the LDL receptor pathway in the liver relates to the use of heterologous LDL to measure total LDL uptake. In rats and hamsters, the liver takes up human LDL at only one fifth the rate of homologous LDL. Indeed, human LDL is taken up by the liver at barely twice the rate of methylated human LDL (a marker of receptor-independent uptake), whereas homologous LDL is transported at ten times the rate of methylated human LDL. Thus, the use of heterologous LDL greatly underestimates the contribution of the liver to whole body LDL catabolism and also greatly underestimates the proportion of hepatic LDL uptake that is mediated by LDL receptors. Although data are limited, the liver also appears to be the major site of LDL catabolism in humans. In one patient with homozygous familial hypercholesterolemia, plasma LDL cholesterol levels fell by 81% and the fractional catabolic rate for LDL increased 2.5-fold following successful transplantation with a normal liver, suggesting that hepatic LDL receptors are responsible for a major fraction of LDL turnover.'' Similarly, LDL binding studies performed on homogenates of tissue samples obtained at the time of elective surgery also suggest that the liver is the major site of expression of LDL receptors. l 7 It should be noted that the LDL uptake rates shown in Figure 2 were measured at a single plasma LDL concentration in animals maintained on a low-fat, low-cholesterol diet. If plasma LDL levels were to increase, for example as a result of an increase in the rate of LDL production, then rates of LDL uptake in each organ would increase in a complex fashion, depending on the kinetic characteristics of the receptor-dependent and -independent LDL transport processes in that tissue, and, as a consequence, the relative contributions of each organ to total body LDL catabolism would change,I8 as discussed later in this review.
CELLULAR SITES OF LOW DENSITY LIPOPROTEIN UPTAKE AND CATABOLISM IN THE LIVER
and several proteins involved in hepatic sterol mctabolism have been shown to be expressed preferentially in periportal or perivenous hepatocytes. For example, in control rats 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is localized largely, if not exclusively, to a few hepatocytes surrounding the portal tract. When animals are treated with lovastatin and cholestyramine, the expression of HMG-CoA reductase in the whole liver increases markedly and under these circumstances abundant HMG-CoA reductase can be identified ~~,~~ are also heterogeneous with in all h e p a t o c y t e ~ .Hepatocytes respect to the expression of 7a-hydroxyla~e.~~ In control rats, 7a-hydroxylase activity is localized primarily to perivenous hepatocytes. When animals are treated with a bile acid sequestrant, 7a-hydroxylase activity in the whole liver increases approximately threefold and this is due largely to induction of 7a-hydroxylase in the remaining midzonal and periportal hepatocytes. Thus, regulation of enzymes involved in sterol metabolism appears to involve regulation of the number of hepatocytes expressing the enzyme as well as regulation of the level of enzyme expression within individual hepatocytes. In the livers of transgenic mice that expressed high levels of the LDL receptor, autoradiographic studies suggested uniform uptake of ' expression of LDL by cells throughout the a c i n ~ s . ~Whether the endogenous LDL receptor is also uniform throughout the acinus is not known.
INTRACELLULAR PATHWAY OF LOW DENSITY LIPOPROTEIN The intracellular pathway of LDL catabolism in hepatocytes has been studied by autoradiography of rat livers following the administration of LDL labeled with "51 or colloidal Shortly after the intravenous administration of labeled LDL or the addition of labeled LDL to the perfusate of isolated livers, LDL binds to receptors distributed diffusely over the microvillus surface of hepatocytes. These receptors, with their bound ligand, then migrate to the base of the microvillus where they are internalized in coated pit regions. The resulting endosomes fuse to form larger endocytic structures and multivesicular bodies. Within these structures, ligand and receptor dissociate and the receptors segregate into tubular appendages, which pinch off and return to the surface. The enlarging vesicular compartment containing the ligand migrates toward the Golgi-lysosome region of the cell, where it fuses with primary lysosomes to form secondary lysosomes. A similar sequence of events has been described in the livers of transgenic mice that express high levels of LDL receptor^.^'
The liver is made up of a variety of cell types, including parenchymal cells (hepatocytes), sinusoidal cells (Kupffer cells, endothelial cells, and fat-storing cells), and cells from the vascular and biliary trees. Among the functionally different cell types identified, hepatocytes constitute about 60% of the cells by number and occupy more than 80% of the total volume KINETIC CHARACTERISTICS OF of the liver. Autoradiographic studies of the liver at early time RECEPTOR-DEPENDENTAND points following the intravenous administration of '251-labeled INDEPENDENT LOW DENSITY LDL indicate that the parenchymal cell is the major cell type LIPOPROTEIN UPTAKE BY THE LIVER involved in the binding and uptake of LDL. l9 The cellular distribution of LDL catabolism has also been examined using As already noted, the liver takes up LDL via receptortechniques to separate parenchymal from nonparenchym~ dependent and -independent pathways, and the rates of uptake cells. When normal or Watanabe heritable hvverlividemic (WHHL) rabbits were injected with h o m o l o g o u l i ~ ~ ' l a b e l e d by these two mechanisms are dependent on the concentration of LDL in the plasma.'* The rate of receptor-dependent LDL with a residualizing marker (I4C-sucrose or 1251-tyramineceiuptake (J,) can be described by classic Michaelis-Menton kilobiose), more than 90% of the label was found in parenchymal netics (Fig. 3). Thus, cells at 24 h o ~ r s , ~again ~ . ~suggesting ' that the parenchymal cell where Jm equals the maximal uptake rate that can be achieved is the major cell type responsible for the terminal catabolism by way of the receptor dependent pathway, K, equals the conof LDL in the liver. In control rabbits, the parenchymal cells centration of LDL in plasma necessary to achieve one half of of the liver account for essentially all (approximately 98%) of this maximal transport rate, and C equals the concentration of the LDL that is taken up via receptor-dependent mechanism^.^^ LDL in the plasma. Jmlargely reflects receptor number but, at Whether all hepatocytes contribute equally to LDL catableast in theory, could also be affected by alterations in receptor olism is not known. Hepatocytes are functionally heterogeneous, depending on their location within the liver l o b ~ l e , ~ ~cycling , ~ ~ time or the distribution of receptors within the cell. Km
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HEPATIC CLEARANCE OF LIPOPROTEINS-SPADY
SEMINARS IN LIVER DISEASE-VOLUME
LDL Production
12, NUMBER 4, 1992
LDL Catabolism
Receptor ....................
[LDLl
-------
[LDLI FIG. 3. Major pathways involved in the production and catabolism of LDL. LDL are formed during the metabolism of VLDL, which, in turn, are secreted by the liver. Thus, the rate of LDL production is dependent on the rate of VLDL secretion by the liver and on the fraction of VLDL that is converted to LDL. LDL is cleared from plasma by receptor-dependent
and -independent transport mechanisms in the various organs of the body. Rates of receptordependent LDL uptake can be described in terms of Michaelis-Menton kinetic parameters where the J m equals the maximal rate of LDL uptake by way of the receptor pathway (a reflection of receptor number) and the Km equals the concentration of LDL necessary to achieve one half of this maximal transport rate (a reflection of receptor affinity). Receptorindeoendent LDL uotake is nonsaturable and can be described in terms of a proportionality constant (P). reflects the affinity of the receptor for the LDL particle in vivo. The affinity of LDL for its receptor could theoretically be altered by changes in the physical-chemical properties of the sinusoidal membranez9 or the LDL particle'0-" or by genetic polymorphisms of the LDL receptor or ap0B100."-'~ The rate of receptor-independent uptake (j,)is directly proportional to C and can be described by the relationship: Ji = PC where P is the proportionality constant of this relationship (Fig. 3). The total rate of LDL uptake is described by the relationship: J, = J, + J, = JmC/K, + C + PC The kinetic parameters for LDL uptake by the liver and other organs have been determined in vivo in the rat and the h a m ~ t e r .The ' ~ values for these parameters were derived from studies in which rates of LDL uptake by individual organs were measured under conditions in which plasma LDL concentrations were acutely raised and maintained at various levels throughout the 4 to 6-hour experimental period. Saturable receptor-dependent LDL uptake could be demonstrated in several tissues, including the liver, endocrine organs, small intestine, spleen, lung, and kidney. When the saturation curves for LDL uptake in these organs were subjected to nonlinear regression analysis, receptor number (as judged by the values for J"') varied widely. in both the rat and hamster, however, approximately 90% of all receptor activity in the body was locali~ed to the liver. In both species, half-maximal rates of LDL transport in the liver were achieved at a plasma LDL-cholesterol concentration of about 90 mgldl. It is not possible to determine experimentally the concentration of LDL at the plasma membrane of hepatocytes in vivo; however, the vascular spaces of the liver are lined by large sinusoids, making it likely that the concentration of LDL in the space of Disse is similar to that in plasma. Thus, it is likely
that the true K , for the hepatic LDL receptor mechanism in vivo is in the range of 90 mgldl. This is quite different from the situation in cultured fibroblasts where half-maximal binding, internalization, and degradation are seen at LDL-cholesterol concentrations of 2 to 3 mgidl, and the receptor pathway is completely saturated at LDL-cholesterol concentrations of 10 mg/dl.5 Although LDL binding kinetics are somewhat more complicated in cultured hepatocytes, K, values for LDL are generally comparable to those observed in cultured fibrob l a s t ~ . ~ ' Why - ' ~ the kinetics of LDL binding and uptake in cultured cells differ so markedly from those in the liver in vivo has not been explained. Whereas only a few organs manifest saturable, receptordependent LDL transport in vivo, all tissues appear to take up LDL by receptor-independent mechanisms. However, since the P values for receptor independent LDL uptake are generally tow, most tissues transport only small amounts of LDL via this pathway at normal plasma LDL con~entrations.'~*'~ When fed a low cholesterol diet, receptor-independent mechanisms account for about 10% of total LDL uptake by the liver in all species for which data are available. Nevertheless, since receptor-independent LDL uptake does not saturate and increases as a linear function of the plasma LDL concentration, the proportion of total LDL uptake mediated by this pathway increases as plasma LDL concentrations rise. This is particularly true under conditions in which the level of hepatic LDL receptor activity has been suppressed by dietary or genetic factors, in which case uptake via the receptor-independent pathway may predominate. The precise mechanisms responsible for receptor-independent uptake are not completely understood. All ceils, including hepatocytes, internalize plasma membrane as uncoated vesicles
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VLDL
and in the process endocytose Receptor independent LDL uptake is probably mediated by such bulk fluid-phase endocytosis. The exact intracellular itinerary of LDL internalized by this process is not known; however, a significant portion of the internalized LDL is delivered to the lysosomal compartment for terminal catabolism. The metabolic consequences of cholesterol made available to the cell as a result of receptordependent and -independent LDL catabolism appear to be similar if not identical. Thus, when equal amounts of LDL cholesterol were delivered to the liver over a 14-hour period, cholesterol synthesis was suppressed to the same extent and cholesteryl ester levels raised to similar levels regardless of whether the LDL was taken up by receptor-dependent or -independent pathways. l4 The WHHL rabbit has been used as a model to examine the contribution of receptor-independent mechanisms to total LDL uptake by individual organs and the whole body in vivo. In WHHL rabbits, four amino acids are deleted from the ligand This results in the prebinding region of the LDL re~eptor.~' mature degradation of the LDL receptor before reaching the cell surface and, as a consequence, functional expression is dramatically decreased by 95%. In WHHL rabbits LDL is taken up into tissues largely, if not entirely, via receptor-independent processes. Overall, the genetic loss of LDL receptors leads to a 75% reduction in the fractional removal of LDL from plasma. There is also a fivefold increase in the rate of LDL as a result of decreased removal of VLDL remn a n t ~ . ~Together, ' this results in a 20-fold increase in plasma LDL concentrations. Is 42 43 Since receptor-independent transport processes do not saturate, rates of receptor-independent LDL uptake by the various tissues of the body also increase 20fold.".42 AS a consequence, most tissues take up LDL-cholesterol at normal or above normal rates despite the absence of LDL receptor activity leading to normal or elevated tissue cholesterol levels and normal or suppressed rates of de novo sterol s y n t h e ~ i s . ~In' ~normal rabbits, receptor-independent mechanisms account for about 12% of total LDL uptake by the liver. In WHHL rabbits, receptor-independent LDL uptake by the liver is increased 20-fold so that the actual mass of LDL-cholesterol taken up by the liver is more than twice as high as in the normal rabbit. As might be anticipated, the increased mass of LDL cholesterol entering the liver in WHHL rabbits results in modest suppression of endogenous sterol synthesis.44Only in the adrenal gland, where receptor-dependent mechanisms normally account for approximately 98% of total LDL uptake, is the actual rate of LDL cholesterol uptake lower in WHHL than in normal animals.
reduction in the rate of de novo cholesterol synthesis. Sterolmediated regulation of LDL receptor number has been demonstrated in a wide variety of cultured cells, including hepatocytes. Interestingly, the LDL receptor pathway in cultured hepatocytes appears to be relatively resistant to regulation by LDL cholesterol. Thus, concentrations of LDL-cholesterol that produce a 10- to 15-fold reduction in LDL receptor number in fibroblasts generally result in only a two- to threefold reduction in LDL receptor number in cultured hepat~cytes.~'.~~.~'-~' The availability of kinetic curves describing LDL transport in normal rats and hamsters has made it possible to evaluate the regulation of receptor-dependent LDL transport by the liver in vivo. In the hamster, receptor-dependent LDL uptake by the liver is clearly regulated by cholesterol a~ailability.~~ Figure 4 shows the changes in hepatic sterol synthesis and receptor-dependent LDL uptake as a function of liver cholesteryl ester levels in hamsters fed varying amounts of cholesterol for 1 month. As is apparent, hepatic sterol synthesis and receptordependent LDL uptake are suppressed as the cholesterol content of the liver increases. However, cholesterol synthesis appears to be more tightly regulated by sterols than is the LDL receptor pathway. Moreover, the time course of regulation of de novo synthesis and receptor-dependent LDL uptake by dietary cholesterol is quite different. Thus, hepatic sterol synthesis is suppressed by over 90% within a few hours of starting a high cholesterol diet. In contrast, suppression of hepatic LDL receptor activity by dietary cholesterol occurs over a period of weeks or months and several days are usually required before In the hamany change in receptor activity can be dete~ted.~? ster, hepatic LDL receptor activity is also regulated by exper-
Dietary cholesterol none @ 0.06 Q 0.12 0 0.24
MECHANISMS OF REGULATION OF RECEPTOR-DEPENDENT LOW DENSITY LIPOPROTEIN UPTAKE BY THE LIVER Sterol-Mediated Regulation Regulation of the LDL receptor pathway has been studied most extensively in cultured human fibroblasts where negative feedback control by cholesterol is the principal form of regulation demonstrated to date.5,45.46 When depleted of cholesterol, these cells increase the number of cell surface LDL receptors and increase the rate of de novo cholesterol synthesis so as to provide the cholesterol needed to maintain membrane synthesis and turnover. Conversely, when cholesterol accumulates in the cell, the number of LDL receptors and the rate of de novo cholesterol synthesis are suppressed. Overall, the addition of saturating levels of LDL cholesterol to fibroblasts that have been grown in lipoprotein-free media results in a 10- to 15fold reduction in the number of LDL receptors and a 50-fold
Hepatic cholesteryl esters (mglg) FIG. 4. Hepatic cholesterol synthesis and LDL receptor activity as a function of hepatic cholesteryl ester levels in hamsters fed varying amounts of cholesterol for 1 month. Rates of cholesterol synthesis were measured in vivo using 13H]water.Rates of LDL transport were measured in vivo using primed infusions of [lZ51]tyraminecellobiose-labeled LDL. Values for receptor activity represent the rates of receptor-dependent LDL uptake in experimental animals a s percentages of the rates of receptor-dependent LDL uptake that would occur in control animals at the same LDL concentration.
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HEPATIC CLEARANCE OF LIPOPROTEINS-SPADY
SEMINARS IN LIVER DISEASE-VOLUME
12, NUMBER 4, 1992
exist. For example, in the rat, hepatic LDL receptor activity is imental conditions that alter net sterol loss from the liver. Thus, not suppressed at liver cholesterol levels associated with blocking the conversion of cholesterol to bile acids by feeding marked suppression of the LDL receptor pathway in other specholic acid leads to suppression of hepatic sterol synthesis and cies, such as hamster or rabbkS4 The basis for such variabilConversely, acceleratLDL receptor activity in the ham~ter.~' ity in sterol-mediated regulation of the LDL receptor pathway ing the conversion of cholesterol to bile acids by the adminisis not known, but presumably represents differences in the tration of bile acid sequestrants results in derepression of heresponsible for sensing the size of the cholesterol ~~ patic cholesterol synthesis and LDL receptor a ~ t i v i t y . " . ~ ~ . mechanisms pool within the hepatocyte and transducing this information to Again, hepatic cholesterol synthesis is regulated much more the nucleus. rapidly and to a greater extent than is hepatic LDL receptor In addition to species differences in responsiveness to diactivity. For example, in the hamster cholestyramine increases etary cholesterol, there is marked variability in the response to hepatic cholesterol synthesis 10- to 20-fold but increases hedietary cholesterol among individuals of the same species.'*-'* patic LDL receptor activity by twofold or l e ~ s . ' ' . ~ ~ . ~ ~ Thus, in response to a given amount of dietary cholesterol, There are no data in humans regarding the regulation of some individuals manifest a marked rise in plasma LDL conreceptor-dependent LDL transport by the liver; however, indicentrations (hyperresponders), others show little or no change rect evidence suggests that the LDL receptor pathway in human -. (hyporesponders), and most show intermediate responses. liver is also regulated by cholesterol availability. LDL binding These differences in response to dietary cholesterol are reprostudies have been carried out on liver samples from gallstone ducible and persist when other dietary and environmental facpatients undergoing elective cholecystectomy. These studies tors are controlled for and therefore are thought to represent demonstrated a significant increase in heparin-sensitive binding genetic variations at gene loci involved in the maintenance of to liver homogenates and a significant reduction in plasma LDL sterol balance or the control of plasma LDL levels. Since inconcentrations in patients treated with bile acid sequestrants or dividual responses to dietary cholesterol are normally distribHMG-CoA reductase inhibitors for 2 to 3 weeks prior to suruted, the genetic effect is thought to be polygenic, that is, gery.56.57 caused by the contribution of several genes with small additive Studies in a variety of cell lines show that sterol-mediated effects. Variability of response to dietary cholesterol occurs in changes in LDL receptor number are generally accompanied all species that have beeiexamined; however, this phenomena by similar changes in receptor mRNA levels, suggesting reghas been investigated most extensively in nonhuman primates ulation at the transcriptional Sterol-mediated reguand rabbits. In general, differences in cholesterol absorption lation of the LDL receptor promoter has been localized to a 10 efficiency or differences in the ability to increase the rate of base pair sequence in the 5'-flanking region of the gene termed bile acid synthesis in response to dietary cholesterol appear to the "sterol regulatory element I" (SRE I).h'-64Point mutations be the major factors differentiating hypo- from hyperrespondwithin the SRE I of the LDL receptor largely prevent the ining animal^.'^-?^ However, the actual genes involved and the duction of transcription that normally occurs in the absence of polymorphisms responsible for variability in the regulation of sterols but do not alter transcription in the presence of sterols. these pathways have yet to be identified. In recent studies in Thus the SRE I of the LDL receptor promoter appears to funchypo- and hyperresponding cynomolgus monkeys, we were tion as a conditional positive element that enhances transcripunable to demonstrate any differences in the regulation of hetion in the absence of sterols and loses its function in the prespatic cholesterol synthesis or receptor-dependent LDL transence of sterols." The sterol-sensitive transacting factor or port that would explain the marked variation in response to factors that interact with the SRE I have yet to be identified. dietary cholesterol in this species (Spady D K , Turley S D, By analogy with the glucocorticoid receptor, such transacting Dietschy J M: Unpublished study). Nevertheless, heritable difproteins might be expected to bind cholesterol (or a metabolite ferences in the regulation of these pathways may contribute to of cholesterol). differences in individual responses to dietary cholesterol in Although cholesterol appears to be the major physiologic other species. regulator of LDL receptor expression, indirect evidence sugA number of mutations and polymorphisms in the LDL ~ ~ than chogests that a polar derivative of ~ h o l e s t e r o l ,rather receptor gene have been identified that may affect the basal lesterol itself, may be responsible for effecting feedback level of receptor expression or the sensitivity of the receptor to r e p r e ~ s i o n67. ~A~ number of sterol binding proteins have been regulation by dietary lipids. Major mutations in the LDL recloned, but the function of these proteins is not known nor is ceptor gene cause elevated plasma LDL concentrations and a it known whether any of these specifically recognize the SRE 1.68-70 Heterozygous disorder called familial hyperch~lesterolemia.~ carriers of the gene produce approximately half the normal Variability of Response to number of LDL receptors, resulting in a twofold elevation of plasma LDL concentrations. Dozens of different mutations of Dietary Cholesterol the LDL receptor gene have been described that lead to the The expression of nonfunctional or dysfunctional re~eptors.~.*O Although dietary cholesterol raises plasma LDL concenfrequency of these mutations, however, is small (about 1 5 0 0 trations, the response to a given load of dietary cholesterol varin the general population) so that they account for only a small ies enormously among different species. In some species, such part of the variability in plasma LDL levels. In addition to the as the hamster, increasing loads of dietary cholesterol produce mutations that give rise to familial hypercholesterolemia, condose-dependent reductions in hepatic LDL receptor activity siderable genetic heterogeneity exists within the normal LDL leading to reciprocal changes in plasma LDL concentration^,^^ r e c e p t ~ r . ~In' , ~ particular, ~ the presence of a PvuII restriction whereas other species, such as the rat, are notoriously resistant site in intron 15 of the LDL receptor gene has been associated to the effects of dietary c h o l e ~ t e r o l . Much ~ ~ ~ ~ of ' the interspewith lower plasma LDL concentrations in populations from cies variability in responsiveness to dietary cholesterol can be Norway,83germ an^,^ and Italy.85Similarly, the presence of a explained by differences in cholesterol absorption efficiency, AvaII restriction site in intron 17 of the LDL receptor gene is differences in the amount of dietary cholesterol that can be associated with lower plasma LDL concentrations in baboon^.^' compensated for by suppressing de novo cholesterol synthesis, The mechanism of these associations is unknown; however, or differences in the ability to increase the rate of conversion since the variable sites are located in introns of the gene, it is of cholesterol to bile salts. In addition, however, differences in presumed that the polymorphisms are in population association the regulation of the LDL receptor mechanism itself probably
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378
HEPATIC CLEARANCE OF LIPOPROTEINS-SPADY
Regulation of Hepatic Low Density Lipoprotein Transport by Dietary Fatty Acids The amount and, more importantly, the type of triglyceride in the diet strongly influences total and LDL cholesterol levels in animals and humans. Thus, triglycerides containing predominantly saturated fatty acids generally raise total and LDL cholesterol levels relative to triglycerides containing predominantly unsaturated fatty acid^.^"' The effect of dietary triglycerides on the processes that control plasma LDL concentrations have been examined in detail in the hamster, an animal model whose response to dietary lipids is similar to that observed in humans. These studies have shown that dietary triglycerides produce their differential effects on circulating LDL levels primarily by altering the level of receptor-dependent LDL transport in the liver."." However, the effects of dietary triglycerides on hepatic LDL receptor activity are complex. Dietary triglycerides contain a heterogeneous mixture of fatty acids that vary in chain length and in the number, position, and configuration (cis or trans) of double bonds. Moreover, an interaction between dietary cholesterol and triglycerides exists whereby small amounts of cholesterol amplify the differential effects of saturated and unsaturated fatty acids.5' In the absence of dietary cholesterol, unsaturated fatty acids modestly increase hepatic LDL receptor activity, whereas saturated fatty acids have little effect. In the presence of dietary cholesterol, saturated fatty acids augment the suppressive activity of cholesterol on the LDL receptor pathway, whereas unsaturated fatty acids partially prevent this effect. Thus, as shown in Figure 5, at any level of dietary cholesterol, hepatic LDL receptor activity is always higher in animals fed unsaturated fatty acids than in animals fed saturated fatty acids. The differential effects of dietary fatty acids are greatest at modest dietary cholesterol intakes where hepatic LDL receptor ac-
Safflower oil CI Coconut oil
b.. --. '-....
-
........................... .......................CI
Dietary Cholesterol (O/O) FIG. 5. Hepatic LDL receptor activity and cholesteryl ester levels in hamsters fed diets supplemented with 20% safflower oil or coconut oil and varying amounts of cholesterol. Rates of LDL transport were measured in vivo using primed infusions of [1z51]tyraminecellobiose-labeled LDL. Val-
ues for receptor activity represent the rates of receptor-dependent LDL uptake in experimental animals a s percentages of the rates of receptor-dependent LDL uptake that would occur in control animals at the same LDL concentration.
tivity may be two- to threefold higher in animals fed unsaturated fatty acids than in animals fed saturated fatty acids. In the hamster, oleate and linoleate are the most active at increasing hepatic LDL receptor activity. The long chain w-3 polyunsaturated fatty acids increase hepatic LDL receptor activity in some species such as the ratv3but have little effect in other species such as the hamster. As illustrated in Figure 6, changes in hepatic LDL receptor activity generally have tittle effect on the absolute mass of LDL taken up by the liver. Since the liver is the major site of LDL catabolism, changes in the rate of LDL uptake by the liver lead to reciprocal changes in the concentration of LDL in plasma. Changes in plasma LDL concentrations, in turn, alter the rate of hepatic LDL uptake according to the kinetics of the receptor-dependent and -independent transport mechanisms. For example, dietary cholesterol and saturated fat markedly suppress hepatic LDL receptor activity. This leads to an increase in circulating LDL levels and in the new steady-state the mass of LDL entering the liver is essentially unchanged; however, receptor-independent processes now account for nearly 50% of total LDL uptake by the liver (compared with about 10% in control animals). On the other hand, an increase in hepatic LDL receptor activity leads to a decline in plasma LDL levels. In the new steady-state the increase in LDL receptor activity allows the liver to transport approximately the same mass of LDL, although this is now accomplished at a much lower plasma LDL concentration.
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with functionally important sequence changes elsewhere in the gene that alter the function of the receptor or the regulation of gene expression. As with the LDL receptor, apoB100 is highly polymorphic and several mutations and polymorphisms of apoBl0O have been identified that alter the affinity of the LDL particle for the LDL receptor and thereby affect receptor-dependent LDL transport. Familial defective apoB 100 is caused by a mutation in the receptor binding domain of ApoB100 that changes an arginine to a glutamine and results in a protein with greatly reduced receptor-binding activity.j3 Heterozygotes for this disorder, which occurs in the general population with a frequency of approximately 1:500, have moderate to marked elevations in plasma LDL level^.^^.*^ In these individuals, autologous LDL (which contains both normal and defective apoB 100) is cleared more slowly from plasma than normal LDLXX and binds to the LDL receptor with reduced affinity.xYeveral additional mutations in the apoB100 gene have been identified that give rise to apoB 100 with altered affinity for the LDL receptor, either in vivo or in ~itro."-'~ Thus, major mutations of the LDL receptor or apoB100, such as occur in familial hypercholesterolemia and familial defective apoB 100, respectively, profoundly affect receptordependent LDL uptake by the liver. The frequency of these mutations is low, however, and they account for only a small fraction of the variability in plasma LDL levels in the population at large. Rather, more subtle polymorphisms of the LDL receptor, apoBlOO and other proteins involved in sensing the size of the cellular pool of cholesterol and mediating feedback repression pobably explain the wide variability in plasma LDL concentrations (and presumably rates of receptor-dependent LDL uptake by the liver) that is seen in affluent populations.
SEMINARS IN LIVER DISEASE-VOLUME
12, NUMBER 4, 1992
0 Coconut Oil ..
Receptor independent
.\\\\.' .\\.,. ., I\\\.
0
40
uptake
80
120
160
1
200
Plasma LDL-Cholesterol Concentration (mgldl) FIG. 6. Hepatic LDL-cholesterol uptake in hamsters fed 20% safflower oil o r coconut oil in the presence o r absence of 0.12% cholesterol. The shaded areas represent the kinetic curves for total (stippled) and receptor-independent (hatched) LDL-cholesterol uptake determined in control animals a s described in the text. Superimposed on these normal kinetic curves are the absolute rates of total LDL-cholesterol uptake in the experimental animals plotted a s a function of the plasma LDL-cholesterol concentration in the same animals.
Although dietary triglycerides containing saturatcd fatty acids generally suppress receptor-dependent LDL uptakc in the liver, it is now clear that not all saturated fatty acids are equally active in this regard. Figure 7 illustrates the results of expcriments in which hepatic receptor-dependent LDL transport was measured in hamsters fed 0.12% cholesterol, 10% olive oil, and 10% of triglycerides made up of the saturated fatty acids 6:O through 18:O." Under these conditions, absorption of the saturated lipid exceeded 95% in all groups. Similarly, cholesterol was absorbed equally well in the seven experimental groups. Of all the saturated fatty acids tested, only lauric (12:0), myristic (14:0), and palmitic (l6:O) acids significantly suppressed hepatic LDL receptor activity and raised plasma LDL concentrations, whereas shorter and longer chain fatty acids had no effect. Similar results have been reported in humans, at least with respect to changes in plasma LDL concentrations. Thus, saturated fatty acids with 10 or fewer carbons have essentially no effect on plasma cholesterol concentrat i o n ~ These . ~ ~ fatty acids are not incorporated into chylomicrons but, rather, pass directly into the portal circulation; whether this accounts for their lack of effect is not known. Similarly, stearic acid also has been shown to have no effect on plasma LDL levels in humans.96Thus, the deleterious effect of saturated fats on hepatic LDL receptor activity is due entirely to their content of lauric, myristic, and palmitic acids. Whereas sterols clearly regulate transcription of the LDL receptor gene leading to changes in receptor number, the mechanism by which dietary fatty acids regulate the LDL receptor pathway has not been established and the available data are conflicting. Studies investigating the effect of dietary triglycerides on hepatic LDL receptor mRNA levels have yielded mixed results. Two groups have shown that saturated and unsaturated triglycerides produce no differential effects on hcpatic LDL receptor mRNA levels in monkeys whether added
In contrast, a third group to low or high cholesterol diets.Y7y8 reported a significant differential effect of saturated and unsaturated triglycerides on hepatic LDL receptor mRNA levels in cholesterol-fed baboons.9y However, in this study the dietary triglycerides produced no differential effects on plasma Plipoprotein or apoB concentrations. Furthermore, in none of these studies were receptor mRNA levels correlated with actual rates of receptor-dependent LDL transport. Recently, we have quantified rates of receptor-dependent LDL transport and LDL receptor mRNA levels in hamsters fed saturated or unsaturated triglycerides. In hamsters fed small amounts of cholesterol (0.05% by weight), maximal rates of receptor-dependent LDL transport in the liver were 2.4-fold higher in animals fed 20% safflower oil than in animals fed the same amount of coconut oil. These changes in maximal rates of LDL transport were accompanied by a similar and highly significant 2.1-fold change in hepatic LDL receptor mRNA levels (Horton J D, Cuthbert J A, and Spady D K: Unpublished observations). Thus, at least under these circumstances, dietary fatty acids produced parallel changes in receptor-dependent LDL transport and receptor mRNA levels, suggesting that fatty acids regulate the LDL receptor pathway at the transcriptional (or at least pretranslational) level. How specific fatty acids might regulate LDL receptor mRNA levels is not known. As already discussed, the major form of regulation identified to date is feedback repression by sterols. Thus, one possibility is that dietary fatty acids produce differential effects on hepatic LDL receptor activity by altering net sterol balance across the liver. Uptake of dietary cholesterol carried in chylomicron remnants and de novo cholesterol synthesis represent the major pathways of net sterol inflow to the liver while secretion of cholesterol into bile, either as such or after conversion to bile acids, represents the major pathway of net sterol loss from the liver (Fig. I). In the hamster, dietary
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,'
12, NO. 4, 1992
Hepatic Clearance of Plasma Low Density Lipoproteins
Complications of atherosclerosis, most notably coronary heart disease and myocardial infarction, are a major cause of morbidity and mortality in Western populations. Of the many risk factors that have been associated with the development of atherosclerosis and coronary heart disease, an elevated concentration of low density lipoproteins (LDLs) in the plasma appears The concentration of LDL to be one of the most irn~0rtant.l.~ in plasma is determined by the rate at which LDL enters the plasma relative to the rate at which LDL is cleared from plasma by the various organs of the body. LDLs circulating in the plasma are formed during the metabolism of triglyceride-rich very low density lipoprotein^^,^ (VLDL), which in turn are secreted by the liver (Fig. 1). The primary function of VLDL is to transport triglyceride from the liver to peripheral sites of utilization and storage. Most of the triglyceride in the VLDL particle is hydrolyzed by lipoprotein lipase, an enzyme located on the surface of endothelial cells in skeletal muscle, heart, and adipose tissue. VLDL are thereby converted to VLDL remnants, a portion of which is rapidly cleared by LDL receptors in the liver, while the remainder are metabolized to LDL, apparently as a result of the continued action of lipoprotein lipase and hepatic triglyceride lipase. In most animal species, the majority of VLDL is rapidly cleared from plasma as VLDL remnants, and plasma LDL concentrations are low.' In humans, however, 80 to 90% of VLDL is ultimately metabolized to LDL particles, which have a much slower t ~ r n o v e r . ~ LDL is taken up into tissues by at least two distinct transport processes. One of these is receptor dependent and involves the interaction of apolipoprotein (apo) BlOO of LDL with specific receptors on the cell surface followed by clustering of these receptors in coated pits and internalization via an endosoma1 In the fibroblast, the LDL receptor is synthesized on membrane-bound ribosomes, glycosylated in the Golgi apparatus, and then inserted into the plasma membrane. Within 10 minutes, LDL receptors gather into coated pits that invaginate to form endocytic vesicle^.^ Multiple endocytic vesicles fuse to form larger endosomes where the LDL particle dissociates from its receptor as a result of a fall in the pH within the endosome. The LDL receptor, along with a number of other receptors, clusters in a portion of the endosomal membrane that pinches off and returns the receptors to the cell surface while the LDL particles are delivered to lysosomes where they are hydrolyzed to amino acids, fatty acids, and cholesterol. The liberated cholesterol is used to support membrane biosynthesis and also serves as a precursor for bile acids and steroid hormones in the liver and endocrine organs, respectively.
From the Department o f Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas.
Reprint requests: Dr. Spady, Department of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235-8887.
A second process, termed "receptor-independent transport," apparently does not involve specific receptors. In contrast to receptor-dependent LDL uptake, receptor-independent transport is nonsaturable and appears not to be regulated. Rates of receptor-independent LDL uptake are generally low; however, since receptor-independent transport increases as a linear function of plasma LDL concentrations, uptake via this pathway can predominate at high plasma LDL levels. Although the LDL receptor pathway was first described in cultured fibroblasts, it is now known that the majority of LDL receptors are located in the liver and that the liver is the major site of LDL catabolism. Thus, the liver plays a central role in controlling plasma LDL concentrations in that it is the source of LDL via VLDL, is involved in the conversion of VLDL to LDL, and is the major site of LDL catabolism. This review will summarize current information regarding the role of the liver in the catabolism of LDL with particular emphasis on the mechanisms whereby dietary and genetic factors interact to control the rate of receptor-dependent LDL uptake by the liver.
CONTRIBUTION OF THE LIVER TO TOTAL BODY LOW DENSITY LIPOPROTEIN CATABOLISM With the development of radiolabeled markers that are retained by tissues after uptake,& it has become possible to measure rates of LDL uptake accurately in all of the major organs of the body in vivo." Total LDL uptake by individual tissues or the whole body can be determined using homologous LDL, and the receptor-independent component can be determined using LDL that has been modified to eliminate completely its interaction with the LDL receptor. This approach is based on the observation that methylation or glucosylation of the lysine residues of apoB 100 completely eliminates binding ~ , ' difference ~ between total and reto the LDL r e c e p t ~ r . ~The ceptor-independent LDL uptake represents the receptor-dependent component of total LDL uptake. Rates of receptor-dependent and independent LDL uptake by individual organs and the whole body have now been determined in several species, including the hamster, rat, mouse, rabbit, dog, and cynomolgus monkey. A similar tissue distribution of LDL uptake has been observed in all of the species studied thus far",14.1s(Spady D K and Dietschy J M: Unpublished observations). When expressed per gram of tissue, rates of total LDL uptake are highest in the liver, small intestine, endocrine organs, and spleen." Rates of LDL uptake in most other tissues are quite low; indeed, LDL uptake is virtually undetectable in the major tissue compartments of the body such as fat, skeletal muscle, skin, and brain. When whole organ weights are taken into account, the liver is clearly the most important site of LDL catabolism, accounting for approxi-
Copyright O 1992 by Thieme Medical Publishers, Inc., 381 Park Avenue South, New York, NY 10016. All rights reserved.
373
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DAVID K. SPADY, M.D.
12, NUMBER 4, 1992
Extrahepatic tissues FIG. 1. Processes involved in the production and catabolism of LDL and their relationship to the major pathways of net sterol input and loss from the liver. Dietary cholesterol and triglycerides enter the body and are transported within chylomicron (CM) particles to peripheral tissues where, after hydrolysis of most of their triglyceride, the particles are converted to chylomicron remnants that are rapidly removed from plasma, primarily by the liver. Similarly, the liver exports cholesterol and triglyceride within VLDL particles. Following loss of triglyceride in peripheral tissues, a portion of VLDL is converted to VLDL remnants, which are rapidly cleared by the liver, while the remainder of VLDL is further metabolized to LDL particles, which are also cleared primarily by the liver. Finally, secretion of cholesterol into bile, either directly or after conversion to bile acids, provides the only significant route for cholesterol elimination from the body. 7~-OHase:7~hydroxylase.
FIG. 2. Rates of receptor-dependent and -independent LDL uptake by individual organs and the whole body. Rates of LDL uptake were measured in hamsters using primed infusions of [125Utyramine cellobiose-labeled homologous LDL (total LDL uptake by receptor-dependent and receptor-independent pathways) and methylated human LDL (the receptor-independent component of total LDL uptake). Rates of LDL uptake are expressed as the micrograms of LDL cholesterol taken up per whole organ per hour per 100 gm body weight. These values were obtained in animals maintained on standard low-cholesterol, low-triglyceride rodent diet. SB: small bowel.
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SEMINARS IN LIVER DISEASE-VOLUME
mately three fourths of total LDL turnover, as illustrated in Figure 2 for data obtained in the hamster. Moreover, the high rate of LDL uptake by the liver is largely receptor dependent. Thus, in animals fed a low cholesterol diet 80 to 90% of total body receptor-dependent LDL transport takes place in the liver. A technical problem that may lead to underestimation of the importance of the LDL receptor pathway in the liver relates to the use of heterologous LDL to measure total LDL uptake. In rats and hamsters, the liver takes up human LDL at only one fifth the rate of homologous LDL. Indeed, human LDL is taken up by the liver at barely twice the rate of methylated human LDL (a marker of receptor-independent uptake), whereas homologous LDL is transported at ten times the rate of methylated human LDL. Thus, the use of heterologous LDL greatly underestimates the contribution of the liver to whole body LDL catabolism and also greatly underestimates the proportion of hepatic LDL uptake that is mediated by LDL receptors. Although data are limited, the liver also appears to be the major site of LDL catabolism in humans. In one patient with homozygous familial hypercholesterolemia, plasma LDL cholesterol levels fell by 81% and the fractional catabolic rate for LDL increased 2.5-fold following successful transplantation with a normal liver, suggesting that hepatic LDL receptors are responsible for a major fraction of LDL turnover.'' Similarly, LDL binding studies performed on homogenates of tissue samples obtained at the time of elective surgery also suggest that the liver is the major site of expression of LDL receptors. l 7 It should be noted that the LDL uptake rates shown in Figure 2 were measured at a single plasma LDL concentration in animals maintained on a low-fat, low-cholesterol diet. If plasma LDL levels were to increase, for example as a result of an increase in the rate of LDL production, then rates of LDL uptake in each organ would increase in a complex fashion, depending on the kinetic characteristics of the receptor-dependent and -independent LDL transport processes in that tissue, and, as a consequence, the relative contributions of each organ to total body LDL catabolism would change,I8 as discussed later in this review.
CELLULAR SITES OF LOW DENSITY LIPOPROTEIN UPTAKE AND CATABOLISM IN THE LIVER
and several proteins involved in hepatic sterol mctabolism have been shown to be expressed preferentially in periportal or perivenous hepatocytes. For example, in control rats 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is localized largely, if not exclusively, to a few hepatocytes surrounding the portal tract. When animals are treated with lovastatin and cholestyramine, the expression of HMG-CoA reductase in the whole liver increases markedly and under these circumstances abundant HMG-CoA reductase can be identified ~~,~~ are also heterogeneous with in all h e p a t o c y t e ~ .Hepatocytes respect to the expression of 7a-hydroxyla~e.~~ In control rats, 7a-hydroxylase activity is localized primarily to perivenous hepatocytes. When animals are treated with a bile acid sequestrant, 7a-hydroxylase activity in the whole liver increases approximately threefold and this is due largely to induction of 7a-hydroxylase in the remaining midzonal and periportal hepatocytes. Thus, regulation of enzymes involved in sterol metabolism appears to involve regulation of the number of hepatocytes expressing the enzyme as well as regulation of the level of enzyme expression within individual hepatocytes. In the livers of transgenic mice that expressed high levels of the LDL receptor, autoradiographic studies suggested uniform uptake of ' expression of LDL by cells throughout the a c i n ~ s . ~Whether the endogenous LDL receptor is also uniform throughout the acinus is not known.
INTRACELLULAR PATHWAY OF LOW DENSITY LIPOPROTEIN The intracellular pathway of LDL catabolism in hepatocytes has been studied by autoradiography of rat livers following the administration of LDL labeled with "51 or colloidal Shortly after the intravenous administration of labeled LDL or the addition of labeled LDL to the perfusate of isolated livers, LDL binds to receptors distributed diffusely over the microvillus surface of hepatocytes. These receptors, with their bound ligand, then migrate to the base of the microvillus where they are internalized in coated pit regions. The resulting endosomes fuse to form larger endocytic structures and multivesicular bodies. Within these structures, ligand and receptor dissociate and the receptors segregate into tubular appendages, which pinch off and return to the surface. The enlarging vesicular compartment containing the ligand migrates toward the Golgi-lysosome region of the cell, where it fuses with primary lysosomes to form secondary lysosomes. A similar sequence of events has been described in the livers of transgenic mice that express high levels of LDL receptor^.^'
The liver is made up of a variety of cell types, including parenchymal cells (hepatocytes), sinusoidal cells (Kupffer cells, endothelial cells, and fat-storing cells), and cells from the vascular and biliary trees. Among the functionally different cell types identified, hepatocytes constitute about 60% of the cells by number and occupy more than 80% of the total volume KINETIC CHARACTERISTICS OF of the liver. Autoradiographic studies of the liver at early time RECEPTOR-DEPENDENTAND points following the intravenous administration of '251-labeled INDEPENDENT LOW DENSITY LDL indicate that the parenchymal cell is the major cell type LIPOPROTEIN UPTAKE BY THE LIVER involved in the binding and uptake of LDL. l9 The cellular distribution of LDL catabolism has also been examined using As already noted, the liver takes up LDL via receptortechniques to separate parenchymal from nonparenchym~ dependent and -independent pathways, and the rates of uptake cells. When normal or Watanabe heritable hvverlividemic (WHHL) rabbits were injected with h o m o l o g o u l i ~ ~ ' l a b e l e d by these two mechanisms are dependent on the concentration of LDL in the plasma.'* The rate of receptor-dependent LDL with a residualizing marker (I4C-sucrose or 1251-tyramineceiuptake (J,) can be described by classic Michaelis-Menton kilobiose), more than 90% of the label was found in parenchymal netics (Fig. 3). Thus, cells at 24 h o ~ r s , ~again ~ . ~suggesting ' that the parenchymal cell where Jm equals the maximal uptake rate that can be achieved is the major cell type responsible for the terminal catabolism by way of the receptor dependent pathway, K, equals the conof LDL in the liver. In control rabbits, the parenchymal cells centration of LDL in plasma necessary to achieve one half of of the liver account for essentially all (approximately 98%) of this maximal transport rate, and C equals the concentration of the LDL that is taken up via receptor-dependent mechanism^.^^ LDL in the plasma. Jmlargely reflects receptor number but, at Whether all hepatocytes contribute equally to LDL catableast in theory, could also be affected by alterations in receptor olism is not known. Hepatocytes are functionally heterogeneous, depending on their location within the liver l o b ~ l e , ~ ~cycling , ~ ~ time or the distribution of receptors within the cell. Km
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HEPATIC CLEARANCE OF LIPOPROTEINS-SPADY
SEMINARS IN LIVER DISEASE-VOLUME
LDL Production
12, NUMBER 4, 1992
LDL Catabolism
Receptor ....................
[LDLl
-------
[LDLI FIG. 3. Major pathways involved in the production and catabolism of LDL. LDL are formed during the metabolism of VLDL, which, in turn, are secreted by the liver. Thus, the rate of LDL production is dependent on the rate of VLDL secretion by the liver and on the fraction of VLDL that is converted to LDL. LDL is cleared from plasma by receptor-dependent
and -independent transport mechanisms in the various organs of the body. Rates of receptordependent LDL uptake can be described in terms of Michaelis-Menton kinetic parameters where the J m equals the maximal rate of LDL uptake by way of the receptor pathway (a reflection of receptor number) and the Km equals the concentration of LDL necessary to achieve one half of this maximal transport rate (a reflection of receptor affinity). Receptorindeoendent LDL uotake is nonsaturable and can be described in terms of a proportionality constant (P). reflects the affinity of the receptor for the LDL particle in vivo. The affinity of LDL for its receptor could theoretically be altered by changes in the physical-chemical properties of the sinusoidal membranez9 or the LDL particle'0-" or by genetic polymorphisms of the LDL receptor or ap0B100."-'~ The rate of receptor-independent uptake (j,)is directly proportional to C and can be described by the relationship: Ji = PC where P is the proportionality constant of this relationship (Fig. 3). The total rate of LDL uptake is described by the relationship: J, = J, + J, = JmC/K, + C + PC The kinetic parameters for LDL uptake by the liver and other organs have been determined in vivo in the rat and the h a m ~ t e r .The ' ~ values for these parameters were derived from studies in which rates of LDL uptake by individual organs were measured under conditions in which plasma LDL concentrations were acutely raised and maintained at various levels throughout the 4 to 6-hour experimental period. Saturable receptor-dependent LDL uptake could be demonstrated in several tissues, including the liver, endocrine organs, small intestine, spleen, lung, and kidney. When the saturation curves for LDL uptake in these organs were subjected to nonlinear regression analysis, receptor number (as judged by the values for J"') varied widely. in both the rat and hamster, however, approximately 90% of all receptor activity in the body was locali~ed to the liver. In both species, half-maximal rates of LDL transport in the liver were achieved at a plasma LDL-cholesterol concentration of about 90 mgldl. It is not possible to determine experimentally the concentration of LDL at the plasma membrane of hepatocytes in vivo; however, the vascular spaces of the liver are lined by large sinusoids, making it likely that the concentration of LDL in the space of Disse is similar to that in plasma. Thus, it is likely
that the true K , for the hepatic LDL receptor mechanism in vivo is in the range of 90 mgldl. This is quite different from the situation in cultured fibroblasts where half-maximal binding, internalization, and degradation are seen at LDL-cholesterol concentrations of 2 to 3 mgidl, and the receptor pathway is completely saturated at LDL-cholesterol concentrations of 10 mg/dl.5 Although LDL binding kinetics are somewhat more complicated in cultured hepatocytes, K, values for LDL are generally comparable to those observed in cultured fibrob l a s t ~ . ~ ' Why - ' ~ the kinetics of LDL binding and uptake in cultured cells differ so markedly from those in the liver in vivo has not been explained. Whereas only a few organs manifest saturable, receptordependent LDL transport in vivo, all tissues appear to take up LDL by receptor-independent mechanisms. However, since the P values for receptor independent LDL uptake are generally tow, most tissues transport only small amounts of LDL via this pathway at normal plasma LDL con~entrations.'~*'~ When fed a low cholesterol diet, receptor-independent mechanisms account for about 10% of total LDL uptake by the liver in all species for which data are available. Nevertheless, since receptor-independent LDL uptake does not saturate and increases as a linear function of the plasma LDL concentration, the proportion of total LDL uptake mediated by this pathway increases as plasma LDL concentrations rise. This is particularly true under conditions in which the level of hepatic LDL receptor activity has been suppressed by dietary or genetic factors, in which case uptake via the receptor-independent pathway may predominate. The precise mechanisms responsible for receptor-independent uptake are not completely understood. All ceils, including hepatocytes, internalize plasma membrane as uncoated vesicles
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VLDL
and in the process endocytose Receptor independent LDL uptake is probably mediated by such bulk fluid-phase endocytosis. The exact intracellular itinerary of LDL internalized by this process is not known; however, a significant portion of the internalized LDL is delivered to the lysosomal compartment for terminal catabolism. The metabolic consequences of cholesterol made available to the cell as a result of receptordependent and -independent LDL catabolism appear to be similar if not identical. Thus, when equal amounts of LDL cholesterol were delivered to the liver over a 14-hour period, cholesterol synthesis was suppressed to the same extent and cholesteryl ester levels raised to similar levels regardless of whether the LDL was taken up by receptor-dependent or -independent pathways. l4 The WHHL rabbit has been used as a model to examine the contribution of receptor-independent mechanisms to total LDL uptake by individual organs and the whole body in vivo. In WHHL rabbits, four amino acids are deleted from the ligand This results in the prebinding region of the LDL re~eptor.~' mature degradation of the LDL receptor before reaching the cell surface and, as a consequence, functional expression is dramatically decreased by 95%. In WHHL rabbits LDL is taken up into tissues largely, if not entirely, via receptor-independent processes. Overall, the genetic loss of LDL receptors leads to a 75% reduction in the fractional removal of LDL from plasma. There is also a fivefold increase in the rate of LDL as a result of decreased removal of VLDL remn a n t ~ . ~Together, ' this results in a 20-fold increase in plasma LDL concentrations. Is 42 43 Since receptor-independent transport processes do not saturate, rates of receptor-independent LDL uptake by the various tissues of the body also increase 20fold.".42 AS a consequence, most tissues take up LDL-cholesterol at normal or above normal rates despite the absence of LDL receptor activity leading to normal or elevated tissue cholesterol levels and normal or suppressed rates of de novo sterol s y n t h e ~ i s . ~In' ~normal rabbits, receptor-independent mechanisms account for about 12% of total LDL uptake by the liver. In WHHL rabbits, receptor-independent LDL uptake by the liver is increased 20-fold so that the actual mass of LDL-cholesterol taken up by the liver is more than twice as high as in the normal rabbit. As might be anticipated, the increased mass of LDL cholesterol entering the liver in WHHL rabbits results in modest suppression of endogenous sterol synthesis.44Only in the adrenal gland, where receptor-dependent mechanisms normally account for approximately 98% of total LDL uptake, is the actual rate of LDL cholesterol uptake lower in WHHL than in normal animals.
reduction in the rate of de novo cholesterol synthesis. Sterolmediated regulation of LDL receptor number has been demonstrated in a wide variety of cultured cells, including hepatocytes. Interestingly, the LDL receptor pathway in cultured hepatocytes appears to be relatively resistant to regulation by LDL cholesterol. Thus, concentrations of LDL-cholesterol that produce a 10- to 15-fold reduction in LDL receptor number in fibroblasts generally result in only a two- to threefold reduction in LDL receptor number in cultured hepat~cytes.~'.~~.~'-~' The availability of kinetic curves describing LDL transport in normal rats and hamsters has made it possible to evaluate the regulation of receptor-dependent LDL transport by the liver in vivo. In the hamster, receptor-dependent LDL uptake by the liver is clearly regulated by cholesterol a~ailability.~~ Figure 4 shows the changes in hepatic sterol synthesis and receptor-dependent LDL uptake as a function of liver cholesteryl ester levels in hamsters fed varying amounts of cholesterol for 1 month. As is apparent, hepatic sterol synthesis and receptordependent LDL uptake are suppressed as the cholesterol content of the liver increases. However, cholesterol synthesis appears to be more tightly regulated by sterols than is the LDL receptor pathway. Moreover, the time course of regulation of de novo synthesis and receptor-dependent LDL uptake by dietary cholesterol is quite different. Thus, hepatic sterol synthesis is suppressed by over 90% within a few hours of starting a high cholesterol diet. In contrast, suppression of hepatic LDL receptor activity by dietary cholesterol occurs over a period of weeks or months and several days are usually required before In the hamany change in receptor activity can be dete~ted.~? ster, hepatic LDL receptor activity is also regulated by exper-
Dietary cholesterol none @ 0.06 Q 0.12 0 0.24
MECHANISMS OF REGULATION OF RECEPTOR-DEPENDENT LOW DENSITY LIPOPROTEIN UPTAKE BY THE LIVER Sterol-Mediated Regulation Regulation of the LDL receptor pathway has been studied most extensively in cultured human fibroblasts where negative feedback control by cholesterol is the principal form of regulation demonstrated to date.5,45.46 When depleted of cholesterol, these cells increase the number of cell surface LDL receptors and increase the rate of de novo cholesterol synthesis so as to provide the cholesterol needed to maintain membrane synthesis and turnover. Conversely, when cholesterol accumulates in the cell, the number of LDL receptors and the rate of de novo cholesterol synthesis are suppressed. Overall, the addition of saturating levels of LDL cholesterol to fibroblasts that have been grown in lipoprotein-free media results in a 10- to 15fold reduction in the number of LDL receptors and a 50-fold
Hepatic cholesteryl esters (mglg) FIG. 4. Hepatic cholesterol synthesis and LDL receptor activity as a function of hepatic cholesteryl ester levels in hamsters fed varying amounts of cholesterol for 1 month. Rates of cholesterol synthesis were measured in vivo using 13H]water.Rates of LDL transport were measured in vivo using primed infusions of [lZ51]tyraminecellobiose-labeled LDL. Values for receptor activity represent the rates of receptor-dependent LDL uptake in experimental animals a s percentages of the rates of receptor-dependent LDL uptake that would occur in control animals at the same LDL concentration.
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HEPATIC CLEARANCE OF LIPOPROTEINS-SPADY
SEMINARS IN LIVER DISEASE-VOLUME
12, NUMBER 4, 1992
exist. For example, in the rat, hepatic LDL receptor activity is imental conditions that alter net sterol loss from the liver. Thus, not suppressed at liver cholesterol levels associated with blocking the conversion of cholesterol to bile acids by feeding marked suppression of the LDL receptor pathway in other specholic acid leads to suppression of hepatic sterol synthesis and cies, such as hamster or rabbkS4 The basis for such variabilConversely, acceleratLDL receptor activity in the ham~ter.~' ity in sterol-mediated regulation of the LDL receptor pathway ing the conversion of cholesterol to bile acids by the adminisis not known, but presumably represents differences in the tration of bile acid sequestrants results in derepression of heresponsible for sensing the size of the cholesterol ~~ patic cholesterol synthesis and LDL receptor a ~ t i v i t y . " . ~ ~ . mechanisms pool within the hepatocyte and transducing this information to Again, hepatic cholesterol synthesis is regulated much more the nucleus. rapidly and to a greater extent than is hepatic LDL receptor In addition to species differences in responsiveness to diactivity. For example, in the hamster cholestyramine increases etary cholesterol, there is marked variability in the response to hepatic cholesterol synthesis 10- to 20-fold but increases hedietary cholesterol among individuals of the same species.'*-'* patic LDL receptor activity by twofold or l e ~ s . ' ' . ~ ~ . ~ ~ Thus, in response to a given amount of dietary cholesterol, There are no data in humans regarding the regulation of some individuals manifest a marked rise in plasma LDL conreceptor-dependent LDL transport by the liver; however, indicentrations (hyperresponders), others show little or no change rect evidence suggests that the LDL receptor pathway in human -. (hyporesponders), and most show intermediate responses. liver is also regulated by cholesterol availability. LDL binding These differences in response to dietary cholesterol are reprostudies have been carried out on liver samples from gallstone ducible and persist when other dietary and environmental facpatients undergoing elective cholecystectomy. These studies tors are controlled for and therefore are thought to represent demonstrated a significant increase in heparin-sensitive binding genetic variations at gene loci involved in the maintenance of to liver homogenates and a significant reduction in plasma LDL sterol balance or the control of plasma LDL levels. Since inconcentrations in patients treated with bile acid sequestrants or dividual responses to dietary cholesterol are normally distribHMG-CoA reductase inhibitors for 2 to 3 weeks prior to suruted, the genetic effect is thought to be polygenic, that is, gery.56.57 caused by the contribution of several genes with small additive Studies in a variety of cell lines show that sterol-mediated effects. Variability of response to dietary cholesterol occurs in changes in LDL receptor number are generally accompanied all species that have beeiexamined; however, this phenomena by similar changes in receptor mRNA levels, suggesting reghas been investigated most extensively in nonhuman primates ulation at the transcriptional Sterol-mediated reguand rabbits. In general, differences in cholesterol absorption lation of the LDL receptor promoter has been localized to a 10 efficiency or differences in the ability to increase the rate of base pair sequence in the 5'-flanking region of the gene termed bile acid synthesis in response to dietary cholesterol appear to the "sterol regulatory element I" (SRE I).h'-64Point mutations be the major factors differentiating hypo- from hyperrespondwithin the SRE I of the LDL receptor largely prevent the ining animal^.'^-?^ However, the actual genes involved and the duction of transcription that normally occurs in the absence of polymorphisms responsible for variability in the regulation of sterols but do not alter transcription in the presence of sterols. these pathways have yet to be identified. In recent studies in Thus the SRE I of the LDL receptor promoter appears to funchypo- and hyperresponding cynomolgus monkeys, we were tion as a conditional positive element that enhances transcripunable to demonstrate any differences in the regulation of hetion in the absence of sterols and loses its function in the prespatic cholesterol synthesis or receptor-dependent LDL transence of sterols." The sterol-sensitive transacting factor or port that would explain the marked variation in response to factors that interact with the SRE I have yet to be identified. dietary cholesterol in this species (Spady D K , Turley S D, By analogy with the glucocorticoid receptor, such transacting Dietschy J M: Unpublished study). Nevertheless, heritable difproteins might be expected to bind cholesterol (or a metabolite ferences in the regulation of these pathways may contribute to of cholesterol). differences in individual responses to dietary cholesterol in Although cholesterol appears to be the major physiologic other species. regulator of LDL receptor expression, indirect evidence sugA number of mutations and polymorphisms in the LDL ~ ~ than chogests that a polar derivative of ~ h o l e s t e r o l ,rather receptor gene have been identified that may affect the basal lesterol itself, may be responsible for effecting feedback level of receptor expression or the sensitivity of the receptor to r e p r e ~ s i o n67. ~A~ number of sterol binding proteins have been regulation by dietary lipids. Major mutations in the LDL recloned, but the function of these proteins is not known nor is ceptor gene cause elevated plasma LDL concentrations and a it known whether any of these specifically recognize the SRE 1.68-70 Heterozygous disorder called familial hyperch~lesterolemia.~ carriers of the gene produce approximately half the normal Variability of Response to number of LDL receptors, resulting in a twofold elevation of plasma LDL concentrations. Dozens of different mutations of Dietary Cholesterol the LDL receptor gene have been described that lead to the The expression of nonfunctional or dysfunctional re~eptors.~.*O Although dietary cholesterol raises plasma LDL concenfrequency of these mutations, however, is small (about 1 5 0 0 trations, the response to a given load of dietary cholesterol varin the general population) so that they account for only a small ies enormously among different species. In some species, such part of the variability in plasma LDL levels. In addition to the as the hamster, increasing loads of dietary cholesterol produce mutations that give rise to familial hypercholesterolemia, condose-dependent reductions in hepatic LDL receptor activity siderable genetic heterogeneity exists within the normal LDL leading to reciprocal changes in plasma LDL concentration^,^^ r e c e p t ~ r . ~In' , ~ particular, ~ the presence of a PvuII restriction whereas other species, such as the rat, are notoriously resistant site in intron 15 of the LDL receptor gene has been associated to the effects of dietary c h o l e ~ t e r o l . Much ~ ~ ~ ~ of ' the interspewith lower plasma LDL concentrations in populations from cies variability in responsiveness to dietary cholesterol can be Norway,83germ an^,^ and Italy.85Similarly, the presence of a explained by differences in cholesterol absorption efficiency, AvaII restriction site in intron 17 of the LDL receptor gene is differences in the amount of dietary cholesterol that can be associated with lower plasma LDL concentrations in baboon^.^' compensated for by suppressing de novo cholesterol synthesis, The mechanism of these associations is unknown; however, or differences in the ability to increase the rate of conversion since the variable sites are located in introns of the gene, it is of cholesterol to bile salts. In addition, however, differences in presumed that the polymorphisms are in population association the regulation of the LDL receptor mechanism itself probably
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378
HEPATIC CLEARANCE OF LIPOPROTEINS-SPADY
Regulation of Hepatic Low Density Lipoprotein Transport by Dietary Fatty Acids The amount and, more importantly, the type of triglyceride in the diet strongly influences total and LDL cholesterol levels in animals and humans. Thus, triglycerides containing predominantly saturated fatty acids generally raise total and LDL cholesterol levels relative to triglycerides containing predominantly unsaturated fatty acid^.^"' The effect of dietary triglycerides on the processes that control plasma LDL concentrations have been examined in detail in the hamster, an animal model whose response to dietary lipids is similar to that observed in humans. These studies have shown that dietary triglycerides produce their differential effects on circulating LDL levels primarily by altering the level of receptor-dependent LDL transport in the liver."." However, the effects of dietary triglycerides on hepatic LDL receptor activity are complex. Dietary triglycerides contain a heterogeneous mixture of fatty acids that vary in chain length and in the number, position, and configuration (cis or trans) of double bonds. Moreover, an interaction between dietary cholesterol and triglycerides exists whereby small amounts of cholesterol amplify the differential effects of saturated and unsaturated fatty acids.5' In the absence of dietary cholesterol, unsaturated fatty acids modestly increase hepatic LDL receptor activity, whereas saturated fatty acids have little effect. In the presence of dietary cholesterol, saturated fatty acids augment the suppressive activity of cholesterol on the LDL receptor pathway, whereas unsaturated fatty acids partially prevent this effect. Thus, as shown in Figure 5, at any level of dietary cholesterol, hepatic LDL receptor activity is always higher in animals fed unsaturated fatty acids than in animals fed saturated fatty acids. The differential effects of dietary fatty acids are greatest at modest dietary cholesterol intakes where hepatic LDL receptor ac-
Safflower oil CI Coconut oil
b.. --. '-....
-
........................... .......................CI
Dietary Cholesterol (O/O) FIG. 5. Hepatic LDL receptor activity and cholesteryl ester levels in hamsters fed diets supplemented with 20% safflower oil or coconut oil and varying amounts of cholesterol. Rates of LDL transport were measured in vivo using primed infusions of [1z51]tyraminecellobiose-labeled LDL. Val-
ues for receptor activity represent the rates of receptor-dependent LDL uptake in experimental animals a s percentages of the rates of receptor-dependent LDL uptake that would occur in control animals at the same LDL concentration.
tivity may be two- to threefold higher in animals fed unsaturated fatty acids than in animals fed saturated fatty acids. In the hamster, oleate and linoleate are the most active at increasing hepatic LDL receptor activity. The long chain w-3 polyunsaturated fatty acids increase hepatic LDL receptor activity in some species such as the ratv3but have little effect in other species such as the hamster. As illustrated in Figure 6, changes in hepatic LDL receptor activity generally have tittle effect on the absolute mass of LDL taken up by the liver. Since the liver is the major site of LDL catabolism, changes in the rate of LDL uptake by the liver lead to reciprocal changes in the concentration of LDL in plasma. Changes in plasma LDL concentrations, in turn, alter the rate of hepatic LDL uptake according to the kinetics of the receptor-dependent and -independent transport mechanisms. For example, dietary cholesterol and saturated fat markedly suppress hepatic LDL receptor activity. This leads to an increase in circulating LDL levels and in the new steady-state the mass of LDL entering the liver is essentially unchanged; however, receptor-independent processes now account for nearly 50% of total LDL uptake by the liver (compared with about 10% in control animals). On the other hand, an increase in hepatic LDL receptor activity leads to a decline in plasma LDL levels. In the new steady-state the increase in LDL receptor activity allows the liver to transport approximately the same mass of LDL, although this is now accomplished at a much lower plasma LDL concentration.
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with functionally important sequence changes elsewhere in the gene that alter the function of the receptor or the regulation of gene expression. As with the LDL receptor, apoB100 is highly polymorphic and several mutations and polymorphisms of apoBl0O have been identified that alter the affinity of the LDL particle for the LDL receptor and thereby affect receptor-dependent LDL transport. Familial defective apoB 100 is caused by a mutation in the receptor binding domain of ApoB100 that changes an arginine to a glutamine and results in a protein with greatly reduced receptor-binding activity.j3 Heterozygotes for this disorder, which occurs in the general population with a frequency of approximately 1:500, have moderate to marked elevations in plasma LDL level^.^^.*^ In these individuals, autologous LDL (which contains both normal and defective apoB 100) is cleared more slowly from plasma than normal LDLXX and binds to the LDL receptor with reduced affinity.xYeveral additional mutations in the apoB100 gene have been identified that give rise to apoB 100 with altered affinity for the LDL receptor, either in vivo or in ~itro."-'~ Thus, major mutations of the LDL receptor or apoB100, such as occur in familial hypercholesterolemia and familial defective apoB 100, respectively, profoundly affect receptordependent LDL uptake by the liver. The frequency of these mutations is low, however, and they account for only a small fraction of the variability in plasma LDL levels in the population at large. Rather, more subtle polymorphisms of the LDL receptor, apoBlOO and other proteins involved in sensing the size of the cellular pool of cholesterol and mediating feedback repression pobably explain the wide variability in plasma LDL concentrations (and presumably rates of receptor-dependent LDL uptake by the liver) that is seen in affluent populations.
SEMINARS IN LIVER DISEASE-VOLUME
12, NUMBER 4, 1992
0 Coconut Oil ..
Receptor independent
.\\\\.' .\\.,. ., I\\\.
0
40
uptake
80
120
160
1
200
Plasma LDL-Cholesterol Concentration (mgldl) FIG. 6. Hepatic LDL-cholesterol uptake in hamsters fed 20% safflower oil o r coconut oil in the presence o r absence of 0.12% cholesterol. The shaded areas represent the kinetic curves for total (stippled) and receptor-independent (hatched) LDL-cholesterol uptake determined in control animals a s described in the text. Superimposed on these normal kinetic curves are the absolute rates of total LDL-cholesterol uptake in the experimental animals plotted a s a function of the plasma LDL-cholesterol concentration in the same animals.
Although dietary triglycerides containing saturatcd fatty acids generally suppress receptor-dependent LDL uptakc in the liver, it is now clear that not all saturated fatty acids are equally active in this regard. Figure 7 illustrates the results of expcriments in which hepatic receptor-dependent LDL transport was measured in hamsters fed 0.12% cholesterol, 10% olive oil, and 10% of triglycerides made up of the saturated fatty acids 6:O through 18:O." Under these conditions, absorption of the saturated lipid exceeded 95% in all groups. Similarly, cholesterol was absorbed equally well in the seven experimental groups. Of all the saturated fatty acids tested, only lauric (12:0), myristic (14:0), and palmitic (l6:O) acids significantly suppressed hepatic LDL receptor activity and raised plasma LDL concentrations, whereas shorter and longer chain fatty acids had no effect. Similar results have been reported in humans, at least with respect to changes in plasma LDL concentrations. Thus, saturated fatty acids with 10 or fewer carbons have essentially no effect on plasma cholesterol concentrat i o n ~ These . ~ ~ fatty acids are not incorporated into chylomicrons but, rather, pass directly into the portal circulation; whether this accounts for their lack of effect is not known. Similarly, stearic acid also has been shown to have no effect on plasma LDL levels in humans.96Thus, the deleterious effect of saturated fats on hepatic LDL receptor activity is due entirely to their content of lauric, myristic, and palmitic acids. Whereas sterols clearly regulate transcription of the LDL receptor gene leading to changes in receptor number, the mechanism by which dietary fatty acids regulate the LDL receptor pathway has not been established and the available data are conflicting. Studies investigating the effect of dietary triglycerides on hepatic LDL receptor mRNA levels have yielded mixed results. Two groups have shown that saturated and unsaturated triglycerides produce no differential effects on hcpatic LDL receptor mRNA levels in monkeys whether added
In contrast, a third group to low or high cholesterol diets.Y7y8 reported a significant differential effect of saturated and unsaturated triglycerides on hepatic LDL receptor mRNA levels in cholesterol-fed baboons.9y However, in this study the dietary triglycerides produced no differential effects on plasma Plipoprotein or apoB concentrations. Furthermore, in none of these studies were receptor mRNA levels correlated with actual rates of receptor-dependent LDL transport. Recently, we have quantified rates of receptor-dependent LDL transport and LDL receptor mRNA levels in hamsters fed saturated or unsaturated triglycerides. In hamsters fed small amounts of cholesterol (0.05% by weight), maximal rates of receptor-dependent LDL transport in the liver were 2.4-fold higher in animals fed 20% safflower oil than in animals fed the same amount of coconut oil. These changes in maximal rates of LDL transport were accompanied by a similar and highly significant 2.1-fold change in hepatic LDL receptor mRNA levels (Horton J D, Cuthbert J A, and Spady D K: Unpublished observations). Thus, at least under these circumstances, dietary fatty acids produced parallel changes in receptor-dependent LDL transport and receptor mRNA levels, suggesting that fatty acids regulate the LDL receptor pathway at the transcriptional (or at least pretranslational) level. How specific fatty acids might regulate LDL receptor mRNA levels is not known. As already discussed, the major form of regulation identified to date is feedback repression by sterols. Thus, one possibility is that dietary fatty acids produce differential effects on hepatic LDL receptor activity by altering net sterol balance across the liver. Uptake of dietary cholesterol carried in chylomicron remnants and de novo cholesterol synthesis represent the major pathways of net sterol inflow to the liver while secretion of cholesterol into bile, either as such or after conversion to bile acids, represents the major pathway of net sterol loss from the liver (Fig. I). In the hamster, dietary
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