Extrahepatic Cells Research Articles

High-density lipoprotein turnover

High-density lipoprotein (HDL) metabolism has been reviewed from information derived from turnover studies in humans. The two major HDL apoproteins AI and AII have different removal rates, reflecting the faster catabolism of HDL 2 than of HDL 3. This is caused by the continual cycle of formation of HDL 2 from HDL 3 and its reversion to HDL 3, in response to the need to transport cholesterol and other lipids from extrahepatic cells and catabolized triglyceride-rich lipoproteins. The conversion of HDL 2 to HDL 3 is mediated through a hepatic lipase. Because this lipase is inhibited by estrogen and stimulated by androgens, women have higher HDL 2 levels than men. The synthesis of apoproteins AI and AII is also higher in women than in men. Nutrition also influences HDL turnover. Carbohydrates increase AI and HDL 2 removal, whereas polyunsaturated fatty acids inhibit synthesis. Vegeterians show high HDL removal rates. Thus low-fat, low-cholesterol diets generally lead to lower HDL levels. Disorders that alter HDL composition (such as alcoholic liver disease or Tangier disease) accelerate HDL removal. Other HDL proteins such as apoproteins C and E show faster turnover rates than AI and AII, since the former exchange with triglyceride-rich lipoproteins and participate in their catabolism. Diminished exchange of apoprotein C from HDL to chylomicrons may be responsible for the diminished catabolism of these particles in type V hyperlipoproteinemia. The unusual turnover characteristics of HDL apoprotein AIV are reviewed, suggesting a dual role for this protein in both triglyceride and cholesterol transport. The striking relationship between very low-density lipoprotein (VLDL) and HDL metabolism is expressed in an inverse association between their respective removal rates. That is, when the VLDL removal rate is slow, as in hypertriglyceridemia, HDL removal is fast. This leads to low HDL levels in hypertriglyceridemia, a combination that is favorable for atherosclerosis. The hypothesis is that in hypertriglyceridemia a high rate of transfer of triglyceride to HDL 3 occurs; this produces triglyceride-rich HDL 2, which is highly susceptible to catabolism by hepatic lipase. This not only depletes the HDL 2 concentration but reduces the capacity of available HDL 3 to participate in reverse cholesterol transport

Interaction of high density lipoprotein with its receptor on cultured fibroblasts and macrophages. Evidence for reversible binding at the cell surface without internalization.

Cultured extrahepatic cells possess a specific high affinity receptor for high density lipoprotein (HDL) that is induced by cholesterol delivery to cells. Current results suggest that HDL receptors on cultured human fibroblasts and mouse peritoneal macrophages promote reversible binding of HDL to the cell surface without internalization of lipoprotein particles. When 125I-HDL3 was bound to cultured cells at 0 degrees C and then warmed to 37 degrees C after removal of unbound lipoprotein, most of the cell surface-bound HDL was released rapidly (t1/2 = 3 min) into the medium without entering a cellular pool that was inaccessible to digestion by trypsin at 0 degrees C. This lack of internalization of HDL was evident under conditions where internalization of 125I-low density lipoprotein and 125I-transferrin were readily detected. When cells were exposed to 125I-HDL3 at 37 degrees C, only a trace amount of iodinated apoprotein remained associated with cells after treatment of cells with trypsin. Fibroblasts treated with medium containing increasing concentrations of cholesterol exhibited a dose-dependent increase in reversible, trypsin-sensitive binding of 125I-HDL3 at 37 degrees C without an attendant increase in trypsin-resistant binding. These results suggest that reversible binding of HDL to its cell-surface receptor without subsequent endocytosis of receptor-HDL complexes is the mechanism by which HDL receptors facilitate cholesterol transport from cells.

Effects of Calcium Antagonists and Adrenergic Antihypertensive Drugs on Plasma Lipids and Cellular Cholesterol Metabolism

Calcium antagonists and antihypertensive alpha-adrenergic and beta-adrenergic drugs may cause changes in plasma lipoprotein levels. Different mechanisms by which these antihypertensive agents effect cellular lipid metabolism have been proposed. The activity of lipoprotein lipase that determines the catabolism of very low density lipoproteins (VLDL) is decreased by the beta-blocker propranolol and increased by alpha 1-antagonists. The plasma cholesterol or low density lipoprotein (LDL) level is inversely associated with the number of LDL receptors. Catecholamines suppress the LDL receptor activity, thus leading to an increase in plasma cholesterol concentration. The calcium antagonist verapamil and the beta-blocker propranolol may increase LDL receptor activity either per se or by its antagonizing effect on the catecholamine action. The metabolism of high density lipoproteins (HDL) may be affected directly by catecholamines, which might increase HDL binding activity, thereby enhancing efflux of cholesterol from cells. Catecholamines inhibit cholesterol biosynthesis in extrahepatic cells. The effects are mediated by alpha 2- and beta 2-adrenergic receptors. Accordingly, the alpha 2-agonists clonidine and alpha-methyldopa mimicked and propranolol opposed the catecholamine action. In contrast, the alpha 1 antagonists indoramin, prazosin, and urapidil had no effect on cholesterol synthesis. The results provide evidence that calcium antagonists and various antihypertensive drugs, depending upon their action on beta- or alpha-adrenergic receptors, affect lipid metabolism differently. The metabolic effect may play a role in atherogenesis and may be of clinical importance when antihypertensive treatment is considered.

Cell cultures of bile duct epithelium and the pathogenesis of biliary atresia.

The pathogenesis of biliary atresia is unknown. The authors describe a technique for culturing extrahepatic bile duct epithelial cells of human and bovine origin in monolayer cell cultures. Light-, electron microscopy and immunohistological studies prove the epithelial nature of the cultured cells. Inoculation of the cells with reovirus 3 showed no destruction; adenovirus 6, herpes simplex and polio virus 1 and 2 destroyed the cells within 24 h. The cells produce a growth factor maintaining the integrity of the cells, even in the absence of serum.

Binding of high density lipoprotein to cultured fibroblasts after chemical alteration of apoprotein amino acid residues.

Cultured extrahepatic cells possess a specific high affinity binding site (receptor) for high density lipoprotein (HDL) that is induced by cholesterol delivery to cells. To characterize the binding recognition site(s) on HDL, the ability of HDL to interact with cultured human fibroblasts was assayed after chemical alteration of specific apoprotein amino acid residues. Reduction and alkylation, acetylation, and cyclohexanedione treatment of HDL3 had little or no effect on its cellular binding. Treatment of HDL3 with tetranitromethane (TNM), however, caused a large dose-dependent decrease in binding, with maximum inhibition at 3 mM. Amino acid analysis of the TNM-treated particles showed specific alteration of tyrosine residues, but sodium dodecyl sulfate-gel electrophoresis demonstrated apoprotein cross-linking coincident with decreased binding. These results suggest that modification of HDL tyrosine residues and/or cross-linking of HDL apoproteins alters the ligand site recognized by the HDL receptor. Gradient gel electrophoresis, molecular sieve chromatography, and electron microscopy showed only minor changes in size distribution and shape of HDL3 particles after treatment with 3 mM TNM, but at higher TNM concentrations, coalescence and aggregation of particles was evident. Treatment of HDL3 with 3 mM TNM affected neither its promotion of the low affinity (receptor-independent) cholesterol efflux from cells nor its ability to accept cholesterol from an albumin suspension, yet promotion of high affinity (receptor-dependent) cholesterol efflux from cells was abolished. The finding that TNM treatment of HDL3 decreases both its receptor binding and its promotion of cholesterol efflux from cells without substantial alteration of its physical properties supports the hypothesis that the HDL receptor functions to facilitate cholesterol transport from cells.

Receptor interactions controlling lipoprotein metabolism.

Lipoprotein receptors play a central role in lipoprotein metabolism and a major role in cholesterol homeostasis. The most completely characterized lipoprotein receptor is the LDL (low density lipoprotein) or apo-B,E(LDL) receptor. The apo-B,E(LDL) receptor is present on both hepatic and extrahepatic cells and is responsible for the metabolism of a major portion of plasma LDL. Binding and internalization of LDL particles by this receptor initiates a series of intracellular events, resulting in the regulation of cellular cholesterol metabolism. In addition to the apo-B on LDL interacting with the apo-B,E(LDL) receptor, the apo-E on apo-E-containing lipoproteins is also capable of interacting and regulating intracellular cholesterol metabolism. The liver has also been shown to contain a second distinct lipoprotein receptor that is specific for apo-E. This receptor has been demonstrated on hepatic membranes from humans, dogs, and swine and is referred to as the apo-E receptor. This receptor may be responsible for the clearance of chylomicron remnants from plasma by the liver and may participate in reverse cholesterol transport. Thus, apo-E is a major determinant in lipoprotein metabolism and cholesterol homeostasis. The receptor binding properties of apo-E are well characterized, and a series of structural variants, several with lipoprotein binding defects, have been identified. Studies of the binding activity of these receptor-defective apo-E variants have helped to define the receptor binding domain of apo-E.(ABSTRACT TRUNCATED AT 250 WORDS)

High-affinity association and degradation of 125I-labelled low density lipoproteins by human hepatocytes in primary culture

Catabolism of homologous low density lipoproteins (LDL) was studied in primary culture of human hepatocytes (HH). The cell association and degradation of 125I-labeled LDL ( 125I-LDL) were curvilinear functions of substrate concentration. Cell association and degradation of 125I-LDL were inhibited by excess unlabeled LDL. Reductive methylation of unlabeled LDL abolished its ability to compete with 125I-LDL for cell association and degradation. Preincubation of HH with unlabeled LDL caused a 63% inhibition of the 125I-LDL degradation. It is concluded that the catabolism of LDL by HH proceeds in part through a receptor-mediated pathway similar to that demonstrated on extrahepatic cells. Human hepatocyte LDL Cell association Degradation High affinity association

Interaction of plasma lipoproteins with human platelets

Human plasma low density lipoproteins (LDL) and high density lipoproteins (HDL) were radioiodinated and their interaction with washed human platelets was assessed. Both ligands were bound by the platelet at 37 degrees C, and an apparent equilibrium was attained within two hours. Minimal binding was observed at 22 degrees C or 4 degrees C. The specificity of these interactions was indicated by the observations that: (a) labeled and nonlabeled lipoproteins interacted with the platelet with the same apparent affinity; (b) nonlabeled lipoproteins inhibited binding, whereas unrelated plasma proteins did not; and (c) the platelet-bound ligands exhibited the appropriate apoprotein chain compositions when analyzed by polyacrylamide gel electrophoresis. Binding of HDL and LDL was found to be independent of the state of platelet activation and did not require divalent ions. Binding of HDL to the platelet was saturable, and a class of sites that maximally bound 1,585 +/- 390 HDL particles, with a dissociation constant of 3.1 X 10(-8) mol/L, was identified. Binding of LDL to the platelet was more complex, but evidence for a class of sites that bound 7,075 +/- 4,800 LDL particles, with a dissociation constant of 4 X 10(-8) mol/L, was found. LDL was a poor inhibitor of 125I-HDL binding to the platelet, whereas HDL was an effective inhibitor of 125I-LDL binding. The capacity of HDL to bind or inhibit LDL binding was not dependent on its apoprotein E content. These results are most readily interpreted in terms of two types of lipoprotein interaction sites on platelets: (1) an HDL binding site that does not bind or interacts poorly with LDL, and (2) an LDL binding site that recognizes or is otherwise altered by HDL. The HDL site may be similar to the HDL receptor expressed by steroidogenic tissues in terms of apoprotein specificity. The LDL site is not the same as the LDL receptor of most extrahepatic cells.

Interaction of Plasma Lipoproteins With Human Platelets

Abstract Human plasma low density lipoproteins (LDL) and high density lipoproteins (HDL) were radioiodinated and their interaction with washed human platelets was assessed. Both ligands were bound by the platelet at 37 degrees C, and an apparent equilibrium was attained within two hours. Minimal binding was observed at 22 degrees C or 4 degrees C. The specificity of these interactions was indicated by the observations that: (a) labeled and nonlabeled lipoproteins interacted with the platelet with the same apparent affinity; (b) nonlabeled lipoproteins inhibited binding, whereas unrelated plasma proteins did not; and (c) the platelet-bound ligands exhibited the appropriate apoprotein chain compositions when analyzed by polyacrylamide gel electrophoresis. Binding of HDL and LDL was found to be independent of the state of platelet activation and did not require divalent ions. Binding of HDL to the platelet was saturable, and a class of sites that maximally bound 1,585 +/- 390 HDL particles, with a dissociation constant of 3.1 X 10(-8) mol/L, was identified. Binding of LDL to the platelet was more complex, but evidence for a class of sites that bound 7,075 +/- 4,800 LDL particles, with a dissociation constant of 4 X 10(- 8) mol/L, was found. LDL was a poor inhibitor of 125I-HDL binding to the platelet, whereas HDL was an effective inhibitor of 125I-LDL binding. The capacity of HDL to bind or inhibit LDL binding was not dependent on its apoprotein E content. These results are most readily interpreted in terms of two types of lipoprotein interaction sites on platelets: (1) an HDL binding site that does not bind or interacts poorly with LDL, and (2) an LDL binding site that recognizes or is otherwise altered by HDL. The HDL site may be similar to the HDL receptor expressed by steroidogenic tissues in terms of apoprotein specificity. The LDL site is not the same as the LDL receptor of most extrahepatic cells.

The metabolism in vitro of human low-density lipoprotein by the human hepatoma cell line Hep G2.

The human hepatoma cell line Hep G2 was studied with respect to metabolism of human low-density lipoprotein (LDL). The Hep G2 cells bind, take up and degrade human LDL with a high-affinity saturable and with a low-affinity non-saturable component. The high-affinity binding possesses a KD of 25 nM-LDL and a maximal amount of binding of about 70 ng of LDL-apoprotein/mg of cell protein. The high-affinity binding, uptake and degradation of LDL by Hep G2 cells is dependent on the extracellular Ca2+ concentration and is down-regulated by the presence of fairly high concentrations of extracellular LDL. Incubation of the Hep G2 cells with LDL results in suppression of the intracellular cholesterol synthesis. It is concluded that the human hepatoma cell line Hep G2 possesses specific LDL receptors similar to the LDL receptors demonstrated on extrahepatic tissue cells.

Regulation of expression of genes for enzymes of the mammalian urea cycle in permanent cell-culture lines of hepatic and non-hepatic origin.

We present here the results of investigations conducted by ourselves and others on the regulation of the expression of genes encoding the enzymes of the mammalian urea cycle as manifest in cultured cells of both hepatic and extrahepatic origin. Upon consideration of the recently discovered discrete non-hepatic arginase genetic locus in man and our consequent hypothesis that the form of arginase thus transcribed in such extrahepatic cells functions principally in providing ornithine for protein anabolism and polyamine biosynthesis, rather than in detoxifying ammonia through urea formation, we have chosen instead to study permanent cell lines that are derived from liver and continue to perform a variety of hepatic functions in culture as experimental models for probing the molecular mechanisms underlying the control of ureagenesis within the mature liver cell. Of two such arginase-positive rat-hepatoma lines, we have characterized extensively in one (H4-II-E-C3) the mode of action of glucocorticoids in augmenting the cellular levels of this enzyme as well as of argininosuccinate synthetase. To this end, we have recently demonstrated that these stimulations are both mediated by binding of the hormones to classical cytoplasmic steroid receptors in a specific and saturable fashion and have thus concluded that the H4-II-E-C3 line will provide a suitable cell culture system for subsequent more detailed experiments from which the information garnered will continue to be relevant to the ureagenic pathway as modulated in the differentiated hepatocyte in vivo.

Effect of catecholamines on sterol synthesis in human mononuclear cells

The effect of adrenaline, noradrenaline, isoproterenol and dibutyryl cyclic AMP on the rate of sterol synthesis was studied in freshly isolated human mononuclear cells. Incubation of cells in a medium containing lipid depleted serum led to a two-fold rise in the rate of sterol synthesis from [14C]acetate. Adrenaline, nor-adrenaline and isoproterenol added in a concentration of 2 X 10(-5) mol/l at zero-time resulted in an inhibition of 30% of this increase in sterol synthesis after 4 h, but had no effect when the hormones were given at 4 h. This indicates that the catecholamines require some time to exert their inhibitory action on sterol synthesis. The action of catecholamines on the incorporation of [14C]acetate into sterols could be mimicked by dibutyryl cyclic AMP. Propranolol added in increasing concentrations to the incubation medium diminished or totally prevented the inhibitory effect of adrenaline, noradrenaline and isoproterenol on sterol synthesis. The results indicate that cholesterol synthesis in extrahepatic human cells, regulated predominantly by low density lipoprotein derived from plasma, is under additional control of catecholamines which may act via cyclic AMP.

培養皮膚線維芽細胞におけるLDLリセプターに対する甲状腺ホルモンの影響

An inherited deficiency of low density lipoprotein (LDL) receptors in familial hypercholesterolemia (FH) is assumed to be the cause of the decreased catabolism of LDL. In contrast, in hyperthyroidism there is an increased rate of LDL catabolism. A 58-year-old female patient with heterozygous FH revealed a normal level of serum cholesterol (193mg/dl) in coexistence of hyperthyroidism. After the therapy of hyperthyroidism with radioiodine the patient's lipid profile revealed an increased concentrations of cholesterol (Whole serum 338, VLDL 68, LDL 199, HDL 42mg/dl). There was a significant inverse correlation between her serum cholesterol levels and her serum thyroxine levels (r=-0.815, p<0.01).Since LDL catabolism in vivo is thought to be mediated largely through the LDL receptor, the role of thyroid hormone in this receptor-mediated degradation of LDL was studied in human skin fibroblasts. Confluent cells were exposed to a medium containing 5% lipoprotein deficient serum prepared from a myxedematous patient by ultracentrifugation for 2 days with or without added triiodothyronine (T3). After 125I-LDL was added, the cells were incubated for 6 hours at 37°C for the determinations of 125I-LDL uptake and degradation. In the cells from 2 normal subjects, LDL uptake and degradation was enhanced 29% by preincubation with T3 (1.0μg/ml). In the cells from 2 heterozygous patients with FH, LDL uptake and degradation was enhanced 23% with T3(1.0μg/ml). While, in the cells from 2 homozygous patients with FH, no effect of T3 was observed. These results suggest that normal serum cholesterol levels in a heterozygous patient with FH result in part from an enhanced degradation of LDL by extrahepatic cells exposed to excess thyroid hormone.

Effects of LDL, HDL and their combination on the endogenous cholesterol synthesis in monocyte-macrophages

It is commonly assumed that the low density lipoprotein (LDL) degradation in extra-hepatic cells proceeds partly by way of the high-affinity LDL uptake process. In addition, it has been suggested that extra-hepatic LDL catabolism proceeds partly by way of preferential uptake and subsequent degradation of LDL by phagocytic scavenger cells. In order to study the effect of LDL and high density lipoprotein (HDL) on cholesterol metabolism in such scavenger cells, cultured human or pig monocytes were incubated with varying amounts of human or pig LDL, HDL and LDL/HDL mixtures, and the incorporation of [ 14C]acetate into cholesterol was measured. The addition of LDL suppressed endogenous cholesterol synthesis. In contrast, the addition of HDL had no effect on endogenous cholesterol synthesis. Incubation of monocytes with LDL/HDL mixtures reduced the total amount of de novo synthesized cholesterol to the same extent as incubation with LDL alone. Our results suggest that in cultured monocyte-macrophages HDL does not influence endogenous cholesterol synthesis, even in the presence of LDL.

Effects of hypothyroidism and cholesterol feeding on the clearance of chylomicron remnants in vivo and by rat hepatocyte monolayers.

The hepatic catabolism of chylomicron remnants in normal rats and in hypothyroid rats which were either normocholesterolaemic or made hypercholesterolaemic by feeding cholesterol and cholic acid was studied in vivo and in hepatocyte monolayers. In vivo, the clearance of injected chylomicron remnants labelled with either cholesteryl- or retinyl ester was delayed in the hypercholesterolaemic hypothyroid rats, but not in normocholesterolaemic hypothyroid rats. Cholesteryl-ester-rich hepatocytes from hypercholesterolaemic hypothyroid rats took up remnants less efficiently than did normal hepatocytes. Hepatocytes from normocholesterolaemic hypothyroid rats had a lower cholesteryl ester content and took up remnant particles to a greater degree than did normal hepatocytes. When normal hepatocytes were incubated with hypercholesterolaemic serum or with mevalonolactone, which increased cell cholesteryl ester content, there was a slight suppression of remnant uptake. Also, addition of triiodothyronine to hepatocyte monolayers suppressed rather than increased uptake of chylomicron remnants in hepatocytes. Thus, the study suggests that chylomicron remnant uptake by the liver is not inhibited by hypothyroidism per se but by the cholesterol accumulation in hepatocytes that is the consequence of cholesterol and bile acid feeding of the rats. Although the cholesteryl ester content in hepatocytes seems to determine remnant uptake, the regulation of uptake does not seem to be as effective as that of the LDL receptor in extrahepatic cells.

Apolipoproteins: Significance and measurement

Plasma lipoproteins are particles containing lipid (cholesterol, cholesteryl ester, triglyceride, phospholipid) and specific proteins (apoproteins). Lipoproteins are generally classified by density. This is a useful operational classification and also one which bears some relationship to function, in that the less dense lipoproteins (chylomicrons), very low density lipoprotein (VLDL), low density lipoprotein (LDL) transport lipid from intestine or liver, whereas high density lipoproteins (HDL), it is thought, play a part in the passage of cholesterol from extrahepatic cells to the liver. The apoproteins serve a number of functions, but the function of many is unknown. Known functions include: a. Structural functions. ApoB is an essential protein for the formation of chylomicrons, VLDL and LDL. ApoA appears essential for HDL formation. b. Coenzyme activity. ApoCTI is a coenzyme for lipoprotein lipase, and apoAI for leeithin:cholesterol acyltransferase ( LCAT). c. Markers, allowing uptake and catabolism of specific lipoproteins. ApoB in LDL is taken up at receptor sites on extrahepatic cells. ApoK may act as a marker for the uptake of ‘remnant particles’ in certain conditions. For the complete definition of a lipoprotein particle it is necessary to define not only the density and lipid composition but also the apoprotein composition. Immunoassays for most of the 14 or so apoproteins have been developed. These pose technical problems peculiar to lipoproteins. a. Antigen exposure. An antibody raised against a delipidated apoprotein may not fully recognise the apoprotein when it forms part of a lipoprotein. Portions of the apoprotein may be masked by lipid. It may be necessary to delipidate or in some other way break up the lipoprotein before assay. b. The mobility of a complex lipoprotein in an electroimmunoassay may be affected by the size of the particle and by the other protein components of the lipoprotein. In an electroimmunoassay standards should be similar and in a similar matrix to the test solution. It may be shown in the future that assays for (say) plasma apoB. apoAl and apoE will allow more accurate prediction of the risk of developing ischaemic heart disease in the individual than the assays now available for cholesterol, triglyceride and HDL-cholcsterol. but this has not yet been demonstrated.

The binding of high density lipoproteins to isolated rat hepatocytes.

The association of 125I-labelled rat high density lipoproteins (125-I-HDL) to suspended rat hepatocytes was studied at 4 degrees C. 125 I-HDL associated to isolated hepatocytes by two processes-one of high and one of low affinity. The cell-association of 125I-HDL exhibited saturation kinetics and was inhibited to varying degrees with both rat and human lipoproteins such as VLDL, LDL and HDL but not by lipoprotein deficient serum or asialo-fetuin. The cell-association of 125I-HDL did not require divalent cations and could be reduced by pronase treatment of the cells. The binding site was clearly different from the receptor for LDL in extrahepatic cells since heparin and apolipoprotein E did not compete and cholesterol ester labelled HDL. The number of binding sites for HDL at 4 degrees C was 2.2 X 10(6) per cell and the association constant (Ka) 8.2 X 10(6) (mol/l)-1. Experiments with HDL labelled with [3H] cholesterol by means of lecithin: cholesterol acyltransferase (LCAT, EC 2.3.1.43) in the cholesterol ester moiety suggested that the same mechanism was responsible for the cell-association of HDL prepared this way.

Granuloma pouch assay for mutagenicity testing.

The Granuloma Pouch Assay (GPA) is an animal model in which mutagenic and carcinogenic effects of a testcompound can be detected in rapidly dividing fibroblasts of a granulation tissue in adult male rats. Growth of this tissue was initiated with a small amount of croton oil at the inside wall of a subcutaneous air pouch on the back of the animals. The test compound can be injected either into the pouch (local) or administered by systemic routes. Alkali labile DNA-lesions, chromosome aberrations, sister chromatid exchanges, point mutations and tumor development in situ were determined. The comparison of mutation frequencies after local and systemic administration of testcompounds, provide an estimation of the pharmacokinetic characteristics and the mutagenic potency of the chemical. The local application route allows the detection of locally active mutagens and of compounds which require activation by P-448 dependent mono-oxygenases. Liver mediated proximate metabolites are detectable when they are transformed into ultimate carcinogens in extrahepatic cells whereas chemicals with a strong organ specific activity are not.

The estradiol-stimulated lipoprotein receptor of rat liver. A binding site that membrane mediates the uptake of rat lipoproteins containing apoproteins B and E.

Hepatic catabolism of lipoproteins containing apolipoproteins B or E is enhanced in rats treated with pharmacologic doses of 17 alpha-ethinyl estradiol. Liver membranes prepared from these rats exhibit an increased number of receptor sites that bind 125I-labeled human low density lipoproteins (LDL) in vitro. In the present studies, this estradiol-stimulated hepatic receptor was shown to recognize the following rat lipoproteins: LDL, very low density lipoproteins obtained from liver perfusates (hepatic VLDL), and VLDL-remnants prepared by intravenous injection of hepatic VLDL into functionally eviscerated rats. The receptor also recognized synthetic lamellar complexes of lecithin and rat apoprotein E as well as canine high density lipoproteins containing apoprotein E (apo E-HDLc). It did not recognize human HDL or rat HDL deficient in apoprotein E. Much smaller amounts of this high affinity binding site were also found on liver membranes from untreated rats, the number of such sites increasing more than 10-fold after the animals were treated with estradiol. Each of the rat lipoproteins recognized by this receptor was taken up more rapidly by perfused livers from estrogen-treated rats. In addition, enrichment of hepatic VLDL with C-apoproteins lowered the ability of these lipoproteins to bind to the estradiol-stimulated receptor and diminished their rate of uptake by the perfused liver of estrogen-treated rats, just as it did in normal rats. The current data indicate that under the influence of pharmacologic doses of estradiol the liver of the rat contains increased amounts of a functional lipoprotein receptor that binds lipoproteins containing apoproteins B and E. This hepatic lipoprotein receptor appears to mediate the uptake and degradation of lipoproteins by the normal liver as well as the liver of estradiol-treated rats. The hepatic receptor bears a close functional resemblance to the LDL receptor previously characterized on extrahepatic cells.

Evolution of the LDL receptor concept-from cultured cells to intact animals.

The initial observations in cultured fibroblasts made six years ago allowed the formulation of a series of hypotheses concerning LDL metabolism in tissues of animals and man. The most important of these hypotheses was that a large fraction of LDL was removed from plasma by a specific receptor-mediated uptake mechanism whose function was to supply cholesterol to extrahepatic cells. This hypothesis is strongly supported by genetic observations in patients with familial hypercholesterolemia and by studies of the four model systems discussed above. These studies by no means solve all of the important questions about LDL metabolism. We still need to know which tissues take up the most LDL; we need to know how much LDL is cleared by the liver and whether this clearance involves the same LDL receptor that operates in extra-hepatic cells; we need to know the mechanism for the clearance of the one-half to two-thirds of LDL that leaves the plasma by receptor-independent pathways; and finally we need to know how an abnormal accumulation of LDL in the plasma leads to the deposition of cholesterol in scavenger cells and produces atherosclerosis.