High density lipoprotein and low density lipoprotein catabolism by human liver and parenchymal and non-parenchymal cells from rat liver.

The capacity of the homogenates from human liver, rat parenchymal cells, rat non-parenchymal cells and total rat liver for the breakdown of human and rat high density lipoprotein (HDL) and human low density lipoprotein (LDL) was determined. Human HDL was catabolized by human liver, in contrast to human LDL, the protein degradation of which was low or absent. Human and rat HDL were catabolized by both the rat parenchymal and non-parenchymal cell homogenates with, on protein base, a 10-times higher activity in the non-parenchymal liver cells. This implies that more than 50% of the total liver capacity for HDL protein degradation is localized in these cell types. Human LDL degradation in the rat could only be detected in the non-parenchymal cell homogenates. These findings are discussed in view of the function of HDL and LDL as carriers for cholesterol.     

Role of parenchymal and non-parenchymal rat liver cells in the uptake of cholesterolester-labeled serum lipoproteins.

Very low density lipoprotein (VLDL)-remnants, prepared by extrahepatic circulation of VLDL, labeled biosynthetically in the cholesterol (ester) moiety, were injected intravenously into rats in order to determine the relative contribution of parenchymal and non-parenchymal liver cells to the hepatic uptake of VLDL-remnant cholesterol (esters). 82.7% of the injected radioactivity is present in liver, measured 30 min after injection. The non-parenchymal liver cells contain 3.1±0.1 times the amount of radioactivity per mg cell protein as compared to parenchymal cells. The hepatic uptake of biosynthetically labeled (screened) low density lipoprotein (LDL) and high density lipoprotein (HDL) cholesterolesters amounts to 26.8% and 24.4% of the injected dose, measured 6 h after injection. The non-parenchymal cells contain 4.3±0.8 and 4.1±0.7 times the amount of radioactivity per mg cell protein as compared to parenchymal cells for LDL and HDL, respectively. It is concluded that in addition to parenchymal cells, the non-parenchymal cells play an important role in the hepatic uptake of cholesterolesters from VLDL-remnants, LDL and HDL.     

Specific targeting of high density lipoproteins to liver hepatocytes by incorporation of a tris-galactoside-terminated cholesterol derivative

A triantennary galactose-terminated cholesterol derivative, N-(tris(beta-D-galactopyranosyloxymethyl) methyl)-N alpha-(4(5-cholesten-3 beta-yloxy)succinyl)glycinamide (Tris-Gal-Chol), which dissolves easily in water, was added to human apolipoprotein E-free high density lipoproteins (HDL) in varying quantities. Incorporation of 5 or 13 micrograms of Tris-Gal-Chol into HDL (20 micrograms of protein) stimulates the liver association of the HDL apoprotein radioactivity 24- and 55-fold, respectively, at 10 min after intravenous injection into rats. The increased interaction of Tris-Gal-Chol HDL with the liver is blocked by preinjection of asialofetuin or N-acetylgalactosamine but not influenced by N-acetylglucosamine. The parenchymal liver cell uptake of HDL is stimulated 42- or 105-fold, respectively, by incorporation of 5 or 13 micrograms of Tris-Gal-Chol into HDL (20 micrograms of protein), while the association with nonparenchymal cells is stimulated only 1.7- or 5-fold. It can be calculated that 98.0% of the Tris-Gal-Chol HDL is associated with parenchymal cells. In contrast, incorporation of 13 micrograms of Tris-Gal-Chol into LDL (20 micrograms of protein) leads to a selective association of LDL with nonparenchymal cells (92.3% of the total liver uptake). It is concluded that Tris-Gal-Chol incorporation into HDL leads to a specific interaction of HDL with the asialoglycoprotein (galactose) receptor on parenchymal cells whereas Tris-Gal-Chol incorporation into LDL leads mainly to an interaction with a galactose receptor from Kupffer cells. Probably this highly selective cellular targeting of LDL and HDL by Tris-Gal-Chol is caused by the difference in size between these lipoproteins. The increased interaction of HDL with the parenchymal cells upon Tris-Gal-Chol incorporation is followed by degradation of the apolipoprotein in the lysosomes. It is concluded that Tris-Gal-Chol incorporation into LDL or HDL leads to a markedly increased catabolism of LDL by way of the Kupffer cells and HDL by parenchymal cells which might be used for lowering serum cholesterol levels. The use of Tris-Gal-Chol might also find application for targeting drugs or other compounds of interest to either Kupffer or parenchymal liver cells.     

Differential abilities of mouse liver parenchymal and nonparenchymal cells in HDL and LDL (native and oxidized) association and cholesterol efflux.

The aim of this study was to quantify the abilities of mouse liver parenchymal and nonparenchymal cells with respect to (i) cholesteryl ester (CE) selective uptake from low-density lipoproteins (LDL), oxidized LDL (OxLDL), and high-density lipoprotein (HDL); and (ii) their free cholesterol efflux to HDL. The preparations of cells were incubated with lipoproteins labelled either in protein with iodine-125 or in CE with 3H-cholesterol oleate, and lipoprotein-protein and lipoprotein-CE associations were measured. The associations of LDL-protein and LDL-CE with nonparenchymal cells were 5- and 2-fold greater, respectively, than with parenchymal cells. However, in terms of CE-selective uptake (CE association minus protein association) both types of cell were equivalent. Similar results were obtained with OxLDL, but both types of cell showed higher abilities in OxLDL-CE than in LDL-CE selective uptake (on average by 3.4-fold). The association of HDL-protein with nonparenchymal cells was 3x that with parenchymal cells; however, nonparenchymal cells associated 45% less HDL-CE. Contrary to parenchymal cells, nonparenchymal cells did not show HDL-CE selective uptake activity. Thus parenchymal cells selectively take CE from the 3 types of lipoproteins, whereas nonparenchymal cells exert this function only on LDL and OxLDL. Efflux was 3.5-fold more important in nonparenchymal than in parenchymal cells.     

The sites of degradation of purified rat low density lipoprotein and high density lipoprotein in the rat

Low density lipoprotein and high density lipoprotein were isolated from rat serum by sequential ultracentrifugation in the density intervals 1.025-1.050 g/ml and 1.125-1.21 g/ml, respectively. The isolated lipoproteins were radioiodinated using ICl. Low density lipoprotein was further purified by concanavalin A affinity chromatography and concentrated by ultracentrifugation. 95% of the purified low density lipoprotein radioactivity was precipitable by tetramethylurea, while only 4% was associated with lipids. The radioiodinated high density lipoprotein was incubated for 1 h at 4 degrees C with unlabelled very low density lipoprotein, followed by reisolation by sequential ultracentrifugation. Only 3% of the radioactivity was associated with lipids and 90% was present on apolipoprotein A-I. The serum decay curves of labelled and subsequently purified rat low and high density lipoprotein, measured over a period of 28 h, clearly exhibited more than one component, in contrast to the monoexponential decay curves of iodinated human low density lipoprotein. The decay curves were not affected by the methods used to purify the LDL and HDL preparations. The catabolic sites of the labelled rat lipoproteins were analyzed in vivo using leupeptin-treated rats. In vivo treatment of rats with leupeptin did not affect the rate of disappearance from serum of intravenously injected labelled rat low density lipoprotein and high density lipoprotein. Leupeptin-dependent accumulation of radioiodine occurred almost exclusively in the liver after intravenous injection of iodinated low density lipoprotein, while both the liver and the kidneys showed leupeptin-dependent accumulation of radioactivity after injection of iodinated high density lipoprotein.     

Scavenger receptor BI mediates the selective uptake of oxidized cholesterol esters by rat liver

High density lipoprotein (HDL) can protect low density lipoprotein (LDL) against oxidation. Oxidized cholesterol esters from LDL can be transferred to HDL and efficiently and selectively removed from the blood circulation by the liver and adrenal in vivo. In the present study, we investigated whether scavenger receptor BI (SR-BI) is responsible for this process. At 30 min after injection, the selective uptake of oxidized cholesterol esters from HDL for liver and adrenal was 2.3- and 2.6-fold higher, respectively, than for native cholesterol esters, whereas other tissues showed no significant difference. The selective uptake of oxidized cholesterol esters from HDL by isolated liver parenchymal cells could be blocked for 75% by oxidized LDL and for 50% by phosphatidylserine liposomes, both of which are known substrates of SR-BI. In vivo uptake of oxidized cholesterol esters from HDL by parenchymal cells decreased by 64 and 81% when rats were treated with estradiol and a high cholesterol diet, respectively, whereas Kupffer cells showed 660 and 475% increases, respectively. These contrasting changes in oxidized cholesterol ester uptake were accompanied by similar contrasting changes in SR-BI expression of parenchymal and Kupffer cells. The rates of SR-BI-mediated selective uptake of oxidized and native cholesterol esters were analyzed in SR-BI-transfected Chinese hamster ovary cells. SR-BI-mediated selective uptake was 3.4-fold higher for oxidized than for native cholesterol esters (30 min of incubation). It is concluded that in addition to the selective uptake of native cholesterol esters, SR-BI is responsible for the highly efficient selective uptake of oxidized cholesterol esters from HDL and thus forms an essential mediator in the HDL-associated protection system for atherogenic oxidized cholesterol esters.     

In vivo cholesteryl ester selective uptake of mildly and standardly oxidized LDL occurs by both parenchymal and nonparenchymal mouse hepatic cells but SR-BI is only responsible for standardly oxidized LDL selective uptake by nonparenchymal cells

In blood circulation, low density lipoproteins (LDL) can undergo modification, such as oxidation, and become key factors in the development of atherosclerosis. Although the liver is the major organ involved in the elimination of oxidized LDL (oxLDL), the identity of the receptor(s) involved remains to be defined. Our work aims to clarify the role of the scavenger receptor class B type I (SR-BI) in the hepatic metabolism of mildly and standardly oxLDL as well as the relative contribution of parenchymal (hepatocytes) and nonparenchymal liver cells with a special emphasis on CE-selective uptake. The association of native LDL and mildly or standardly oxLDL labeled either in proteins or in cholesteryl esters (CE) was measured on primary cultures of mouse hepatocytes from normal and SR-BI knock-out (KO) mice. These in vitro assays demonstrated that hepatocytes are able to mediate CE-selective uptake from both LDL and oxLDL and that SR-BI KO hepatocytes have a 60% reduced ability to selectively take CE from LDL but not towards mildly or standardly oxLDL. When lipoproteins were injected in the mouse inferior vena cava, parenchymal and nonparenchymal liver cells accumulated more CE than proteins from native, mildly and standardly oxLDL, indicating that selective uptake of CE from these lipoproteins occurs in vivo in these two cell types. The parenchymal cells contribute near 90% of the LDL-CE selective uptake and SR-BI for 60% of this pathway. Nonparenchymal cells capture mainly standardly oxLDL while parenchymal and nonparenchymal cells equally take up mildly oxLDL. An 82% reduction of standardly oxLDL-CE selective uptake by the nonparenchymal cells of SR-BI KO mice allowed emphasizing the contribution of SR-BI in hepatic metabolism of standardly oxLDL. However, SR-BI is not responsible for mildly oxLDL metabolism. Thus, SR-BI is involved in LDL- and standardly oxLDL-CE selective uptake in parenchymal and nonparenchymal cells, respectively.     

Role of liver endothelial and Kupffer cells in clearing low density lipoprotein from blood in hypercholesterolemic rabbits.

The role of liver endothelial and Kupffer cells in the hepatic uptake of cholesterol-rich low density lipoprotein (LDL) was studied in rabbits fed a diet containing 2% (w/w) cholesterol for 3 weeks. 125I-labeled tyramine cellobiose-labeled cholesterol-rich LDL was injected intravenously into rabbits, and parenchymal and nonparenchymal liver cells were isolated 24 h after injection. The hepatic uptake was 9 +/- 3% of injected dose in cholesterol-fed rabbits 24 h after injection, as compared to 36 +/- 9% in control-fed rabbits (n = 6 in each group; significant difference, P less than 0.005). Endothelial and Kupffer cells took up 2.7 +/- 0.5% and 1.2 +/- 0.8% of injected dose in the hypercholesterolemic rabbits, as compared to 1.9 +/- 0.8% and 0.8 +/- 0.3% in control animals. The amount accounted for by the parenchymal cells was markedly reduced in the cholesterol-fed rabbits to 7.3 +/- 2.7% of injected dose, as compared to 32.8 +/- 7.6% in controls (P less than 0.02). On a per cell basis, the nonparenchymal cells of cholesterol-fed rabbits took up as much LDL as the parenchymal cells (0.6 +/- 0.2, 0.7 +/- 0.1, and 0.6 +/- 0.4% of injected dose per 10(9) parenchymal, endothelial, and Kupffer cells, respectively). This is in marked contrast to the control animals, in which parenchymal cells took up about 6 times more LDL per cell than endothelial and Kupffer cells (3.2 +/- 0.9, 0.7 +/- 0.3, and 0.5 +/- 0.1% of injected dose per 10(9) cells). Thus, 30% of the hepatic uptake of LDL in the cholesterol-fed rabbits took place in nonparenchymal cells, as compared to 6% in controls. Consistent with these data, the concentrations of cholesteryl ester in endothelial and Kupffer cells in rabbits fed the high cholesterol diet were about twofold higher than in parenchymal cells (428 +/- 74 and 508 +/- 125 micrograms/mg protein, respectively, vs. 221 +/- 24 micrograms/mg protein in parenchymal cells). In contrast to cells from normal rabbits, Kupffer and endothelial cells from cholesterol-fed rabbits accumulated significant amounts of Oil Red O-positive material (neutral lipids). Electron microscopic examination of these cells in situ as well as in culture revealed numerous intracellular lipid droplets. Slot blot hybridization of RNA from liver parenchymal, endothelial, and Kupffer cells showed that cholesterol feeding reduced the level of mRNA specific for the apoB,E receptor to a small and insignificant extent in all three cell types (to 70-80% of that observed in control animals).(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of the selective uptake of high density lipoprotein-associated cholesteryl esters by human fibroblasts and Hep G2 hepatoma cells

We have previously shown that the liver and steroidogenic tissues of rats in vivo and a wider range of cells in vitro, including human cells, selectively take up high density lipoprotein (HDL) cholesteryl esters without parallel uptake of HDL particles. This process is regulated in tissues of rats and in cultured rat cells according to their cholesterol status. In the present study, we examined regulation of HDL selective uptake in cultured human fibroblasts and Hep G2 hepatoma cells. The cholesterol content of these cells was modified by a 20-hr incubation with either low density lipoprotein (LDL) or free cholesterol. Uptake of HDL components was examined in a subsequent 4-6-hr assay using intracellularly trapped tracers: 125I-labeled N-methyl-tyramine-cellobiose-apoA-I (125I-NMTC-apoA-I) to trace apoA-I, and [3H]cholesteryl oleyl ether to trace cholesteryl esters. In the case of fibroblasts, pretreatment with either LDL or free cholesterol resulted in decreased selective uptake (total [3H]cholesteryl ether uptake minus that due to particle uptake as measured by 125I-NMTC-apoA-I). In contrast, HDL particle uptake increased with either form of cholesterol loading. The amount of HDL that was reversibly cell-associated (bound) was increased by prior exposure to free cholesterol, but was decreased by prior exposure to LDL. In the case of Hep G2 cells, exposure to free cholesterol only slightly increased HDL particle uptake; selective uptake decreased after both forms of cholesterol loading, and reversibly bound HDL increased after exposure to free cholesterol, but either did not change or decreased after exposure to LDL. It was excluded that either LDL carried over into the HDL uptake assay or that products secreted by the cultured cells influenced these results. Thus, selective uptake by cells of both hepatic and extrahepatic origin was down-regulated by cholesterol loading, under which conditions HDL particle uptake increased. Total HDL binding was not directly correlated with either the rate of selective uptake or the rate of HDL particle uptake or the cholesterol status of the cells, suggesting more than one type of HDL binding site.     

Low and high density lipoprotein metabolism in primary cultures of hepatic cells from normal and apolipoprotein E knockout mice.

Apolipoprotein E (apoE) plays a major role in lipoprotein metabolism by mediating the binding of apoE-containing lipoproteins to receptors. The role of hepatic apoE in the catabolism of apoE-free lipoproteins such as low density lipoprotein (LDL) and high density lipoprotein-3 (HDL(3)) is however, unclear. We analyzed the importance of hepatic apoE by comparing human LDL and HDL(3) metabolism in primary cultures of hepatic cells from control C57BL/6J and apoE knockout (KO) mice. Binding analysis showed that the maximal binding capacity (Bmax) of LDL, but not of HDL(3), is increased by twofold in the absence of apoE synthesis/secretion. Compared to control hepatic cells, LDL and HDL(3) holoparticle uptake by apoE KO hepatic cells, as monitored by protein degradation, is reduced by 54 and 77%, respectively. Cleavage of heparan sulfate proteoglycans (HSPG) by treatment with heparinase I reduces LDL association by 21% in control hepatic cells. Thus, HSPG alone or a hepatic apoE-HSPG complex is partially involved in LDL association with mouse hepatic cells. In apoE KO, but not in normal hepatic cells, the same treatment increases LDL uptake/degradation by 2.4-fold suggesting that in normal hepatic cells, hepatic apoE increases LDL degradation by masking apoB-100 binding sites on proteoglycans. Cholesteryl ester (CE) association and CE selective uptake (CE/protein association ratio) from LDL and HDL(3) by mouse hepatic cells were not affected by the absence of apoE expression. We also show that 69 and 72% of LDL-CE hydrolysis in control and apoE KO hepatic cells, respectively, is sensitive to chloroquine revealing the importance of a pathway linked to lysosomes. In contrast, HDL(3)-CE hydrolysis is only mediated by a nonlysosomal pathway in both control and apoE KO hepatic cells. Overall, our results indicate that hepatic apoE increases the holoparticle uptake pathway of LDL and HDL(3) by mouse hepatic cells, that HSPG devoid of apoE favors LDL binding/association but impairs LDL uptake/degradation and that apoE plays no significant role in CE selective uptake from either human LDL or HDL(3) lipoproteins.     

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.     

Receptor-mediated uptake of low density lipoprotein stimulates bile acid synthesis by cultured rat hepatocytes

The cellular mechanisms responsible for the lipoprotein-mediated stimulation of bile acid synthesis in cultured rat hepatocytes were investigated. Adding 280 micrograms/ml of cholesterol in the form of human or rat low density lipoprotein (LDL) to the culture medium increased bile acid synthesis by 1.8- and 1.6-fold, respectively. As a result of the uptake of LDL, the synthesis of [14C]cholesterol from [2-14C]acetate was decreased and cellular cholesteryl ester mass was increased. Further studies demonstrated that rat apoE-free LDL and apoE-rich high density lipoprotein (HDL) both stimulated bile acid synthesis 1.5-fold, as well as inhibited the formation of [14C]cholesterol from [2-14C]acetate. Reductive methylation of LDL blocked the inhibition of cholesterol synthesis, as well as the stimulation of bile acid synthesis, suggesting that these processes require receptor-mediated uptake. To identify the receptors responsible, competitive binding studies using 125I-labeled apoE-free LDL and 125I-labeled apoE-rich HDL were performed. Both apoE-free LDL and apoE-rich HDL displayed an equal ability to compete for binding of the other, suggesting that a receptor or a group of receptors that recognizes both apolipoproteins is involved. Additional studies show that hepatocytes from cholestyramine-treated rats displayed 2.2- and 3.4-fold increases in the binding of apoE-free LDL and apoE-rich HDL, respectively. These data show for the first time that receptor-mediated uptake of LDL by the liver is intimately linked to processes activating bile acid synthesis.     

Uptake of lactosylated low-density lipoprotein by galactose-specific receptors in rat liver.

The liver contains two types of galactose receptors, specific for Kupffer and parenchymal cells respectively. These receptors are only expressed in the liver, and therefore are attractive targets for the specific delivery of drugs. We provided low-density lipoprotein (LDL), a particle with a diameter of 23 nm in which a variety of drugs can be incorporated, with terminal galactose residues by lactosylation. Radioiodinated LDL, lactosylated to various extents (60-400 mol of lactose/ mol of LDL), was injected into rats. The plasma clearance and hepatic uptake of radioactivity were correlated with the extent of lactosylation. Highly lactosylated LDL (greater than 300 lactose/LDL) is completely cleared from the blood by liver within 10 min. Pre-injection with N-acetylgalactosamine blocks liver uptake, which indicates that the hepatic recognition sites are galactose-specific. The hepatic uptake occurs mainly by parenchymal and Kupffer cells. At a low degree of lactosylation, approx. 60 lactose/LDL, the specific uptake (ng/mg of cell protein) is 28 times higher in Kupffer cells than in parenchymal cells. However, because of their much larger mass, parenchymal cells are the main site of uptake. At high degrees of lactosylation (greater than 300 lactose/LDL), the specific uptake in Kupffer cells is 70-95 times that in parenchymal cells. Under these conditions, Kupffer cells are, despite their much smaller mass, the main site of uptake. Thus not only the size but also the surface density of galactose on lactosylated LDL is important for the balance of uptake between Kupffer and parenchymal cells. This knowledge should allow us to design particulate galactose-bearing carriers for the rapid transport of various drugs to either parenchymal cells or Kupffer cells.     

Specific binding and uptake of apolipoprotein E-free high density lipoproteins by cultured liver parenchymal cells of copper-deficient rats

The influence of copper deficiency on the binding and uptake of apolipoprotein E-free high density lipoprotein (apo E-free HDL) in cultured rat hepatic parenchymal cells was examined in this study. Male weanling Sprague-Dawley rats were randomly divided into two treatments, a Cu-adequate (7.33 mg Cu/kg diet) or a Cu-deficient (1.04 mg Cu/kg diet) group. After 7 weeks, plasma apo E-free HDL were isolated by a combination of ultracentrifugation, gel filtration, and heparin-Sepharose affinity chromatography. Parenchymal cells were isolated from collagenase perfused liver of Cu-deficient and adequate rats and cultured for 16 hours at 37 degrees C prior to incubation with iodinated apo E-free HDL from the same treatment group. Cells were incubated with 5 microg/ml(125) I-apo E-free HDL for 2, 6, or 12 hours in the presence or absence of 200 microg/ml (40-fold) excess unlabeled apo E-free HDL. Increases in specific binding at 4 degrees C and specific cell-associated uptake at 37 degrees C as a function of time were observed with cells and HDL from Cu-deficient rats. Cells were also incubated for 6 hours with 8 concentrations of (125)I-apo E-free HDL in the presence or absence of excess unlabeled HDL. Although no significant increase in specific binding was detected at 4 degrees C as a function of ligand concentration, the response tended to be higher at 5 to 15 microg HDL/ml for the Cu-deficient treatment. However, at 37 degrees C the specific cell-associated uptake was increased markedly with cells and HDL from Cu-deficient rats. The observed increases in HDL binding and uptake indicate that these processes may be enhanced in Cu-deficient rats. These data are also consistent with recent in vivo results which indicate that plasma clearance and tissue uptake of HDL are increased in Cu-deficient rats.     

Uptake and processing of remnants of chylomicrons and very low density lipoproteins by rat liver

In the rat, chylomicron remnants and very low density lipoprotein (VLDL) remnants are taken up into the liver by high affinity processes and appear to undergo degradation by lysosomes. The relationship of this catabolic process to the known pathways of uptake and degradation of low density lipoproteins (LDL) and the involvement of nonparenchymal cells are addressed in these studies. We have utilized both light and electron microscopic radioautography to determine whether the pathway of intracellular transport and catabolism resembles that established for LDL in hepatocytes. Radioiodinated plasma VLDL remnants and lymph chylomicron remnants were injected into femoral veins of rats and the livers were fixed by perfusion 3 to 30 minutes later. Quantitative light microscopic radioautography showed little or no accumulation of grains over Kupffer cells. Electromicroscopic radioautography confirmed these observations and, in addition, demonstrated that very few grains were associated with endothelial cells. The processing of the remnant particles closely resembled that of LDL. Following an initial association of grains with the parenchymal cell plasma membrane, frequently in regions in close proximity to clathrin-coated endocytic pits, the grains were found in endocytic vesicles just beneath the plasma membrane. By 15 minutes the grains were found over multivesicular bodies located in the Golgi-lysosome region of the cell. Thirty minutes after injection, radioautographic grains began to be associated with secondary lysosomes. These data indicate no significant role for nonparenchymal cells in the internalization and subsequent degradation of triglyceride-rich lipoproteins, and provide evidence that the processing of remnants as well as LDL follows the classical pathway of receptor-mediated endocytosis.     

Hepatic processing of the cholesteryl ester from low density lipoprotein in the rat

Human low density lipoprotein (LDL), radiolabeled in the cholesteryl ester moiety, was injected into estrogen-treated and -untreated rats. The hepatic and extrahepatic distribution and biliary secretion of [3H]cholesteryl esters were determined at various times after injection. In order to follow the intrahepatic metabolism of the cholesteryl esters of LDL in vivo, the liver was subfractioned into parenchymal and Kupffer cells by a low temperature cell isolation procedure. In control rats, the LDL cholesteryl esters were mainly taken up by the Kupffer cells. After uptake, the [3H]cholesteryl esters are rapidly hydrolyzed, followed by release of [3H]cholesterol from the cells to other sites in the body. Up to 24 h after injection of LDL, only 9% of the radioactivity appeared in the bile, whereas after 72 h, this value was 30%. Hepatic and especially the parenchymal cell uptake of [3H]cholesteryl esters from LDL was strongly increased upon 17 alpha-ethinylestradiol treatment (3 days, 5 mg/kg). After rapid hydrolysis of the esters, [3H]cholesterol was both secreted into bile (28% of the injected dose in the first 24 h) as well as stored inside the cells as re-esterified cholesterol ester. It is concluded that uptake of human LDL by the liver in untreated rats is not efficiently coupled to biliary secretion of cholesterol (derivatives), which might be due to the anatomical localization of the principal uptake site, the Kupffer cells. In contrast, uptake of LDL cholesterol ester by liver hepatocytes is tightly coupled to bile excretion. The Kupffer cell uptake of LDL might be necessary in order to convert LDL cholesterol (esters) into a less toxic form. This activity can be functional in animals with low receptor activity on hepatocytes, as observed in untreated rats, or after diet-induced down-regulation of hepatocyte LDL receptors in other animals.     

Nitrosylated high density lipoprotein is recognized by a scavenger receptor in rat liver

In order to assess the presence of specific recognition sites for high density lipoprotein (HDL) in vivo, HDL was nitrosylated with tetranitromethane and the decay and liver uptake were compared with that of native HDL. The association of intravenously injected nitrosylated HDL (TNM-HDL) with liver was greatly increased as compared to native HDL. Using a cold cell isolation method, it became evident that the liver endothelial cells were responsible for the increased uptake of the modified HDL. The involvement of the endothelial cells in the uptake of TNM-HDL from the circulation could also be demonstrated morphologically by using the fluorescent dye dioctadecyl-tetramethyl-indocarbocyanine perchlorate (Dil) to label HDL. In vitro competition studies with isolated liver endothelial cells indicated that unlabeled modified HDL and acetylated LDL displaced iodine-labeled TNM-HDL, while no competition was seen with LDL and a slight displacement was seen with unlabeled native HDL. Nonlipoprotein competitors of the scavenger receptor such as fucoidin and polyinosinic acid blocked the interaction of TNM-HDL with the liver endothelial cells. Also the degradation of TNM-HDL was blocked by low concentrations of chloroquine. It can be concluded that a scavenger receptor on liver endothelial cells is involved in the clearance of tetranitromethane-modified HDL, which excludes the possibility of using TNM-HDL in vivo to assess the non-receptor-dependent uptake of HDL. The use of nitrosylated HDL in vitro as a low affinity control is limited to cell types that do not possess scavenger receptors, because cell types with scavenger receptors will recognize and internalize TNM-HDL by a high affinity scavenger pathway.     

The effects of dietary fish oil on hepatic high density and low density lipoprotein receptor activities in the rat

Rats were fed either a standard ration diet or that diet supplemented with 8% by wt of a marine fish oil or safflower oil. After 10 days, plasma triacylglycerols, total cholesterol, high density lipoprotein (HDL) cholesterol, hepatic cholesterol and fatty acid synthesis and hepatic low density lipoprotein (LDL) receptor activity were significantly depressed while HDL receptor activity was significantly increased in rats fed fish oil. Fish oil-induced effects on cholesterol metabolism in the rat therefore include reciprocal changes in the activities of hepatic LDL and HDL receptors.     

Role of low density lipoprotein receptor-dependent and -independent sites in binding and uptake of chylomicron remnants in rat liver

The role of the low density lipoprotein (LDL) receptor in the binding of chylomicron remnants to liver membranes and in their uptake by hepatocytes was assessed using a monospecific polyclonal antibody to the LDL receptor of the rat liver. The anti-LDL receptor antibody inhibited the binding and uptake of chylomicron remnants and LDL by the poorly differentiated rat hepatoma cell HTC 7288C as completely as did unlabeled lipoproteins. The antireceptor antibody, however, decreased binding of chylomicron remnants to liver membranes from normal rats by only about 10%. This was true for intact membranes and for solubilized reconstituted membranes and with both a crude membrane fraction as well as with purified sinusoidal membranes. Further, complete removal of the LDL receptor from solubilized membranes by immunoprecipitation with antireceptor antibody only decreased remnant binding to the reconstituted supernatant by 10% compared to solubilized, nonimmunoprecipitated membranes. Treatment of rats with ethinyl estradiol induced an increase in remnant binding by liver membranes. All of the increased binding could be inhibited by the antireceptor antibody. The LDL receptor-independent remnant binding site was not EDTA sensitive and was not affected by ethinyl estradiol treatment. LDL receptor-independent remnant binding was competed for by beta-VLDL = HDLc greater than rat LDL greater than human LDL (where VLDL is very low density lipoprotein, and HDL is high density lipoprotein). There was weak and incomplete competition by apoE-free HDL, probably due to removal of apoE from the remnant. The LDL receptor-independent remnant-binding site was also present in membranes prepared from isolated hepatocytes and had the same characteristics as the site on membranes prepared from whole liver. In contrast, when chylomicron remnants were incubated with a primary culture of rat hepatocytes, the anti-LDL receptor antibody prevented specific cell association by 84% and degradation of chylomicron remnants completely. Based on these studies, we conclude that although binding of chylomicron remnants to liver cell membranes is not dependent on the LDL receptor, their intact uptake by hepatocytes is.     

Characterization of low density and high density lipoprotein receptors in the rat corpus luteum and regulation by gonadotropin

Freshly prepared plasma membranes from rat corpora lutea were examined for the presence of low density lipoprotein (LDL) and high density lipoprotein (HDL) receptors by determining the specific binding of 125I-LDL and 125I-HDL. These membranes have two types of binding site for 125I-LDL, one with high affinity (Kd = 7.7 micrograms of LDL protein/ml), the other with low affinity (Kd = 213 micrograms of LDL protein/ml) and one type of binding site for 125I-HDL with Kd = 17.8 micrograms of HDL protein/ml. LDL receptor is sensitive to pronase and trypsin; HDL receptor, however, is resistant. The binding reaction was further characterized with respect to effect of time and temperature of incubation, requirement of divalent metal ion, influence of ionic strength, and binding specificity. In vivo pretreatment of rats with human choriogonadotropin (hCG) resulted in induction of both LDL and HDL receptors in a dose- and time-dependent manner when compared with saline-injected controls. The induction of lipoprotein receptors by hCG treatment is target organ-specific since the increase was seen only in the ovarian tissue. Membranes prepared from liver, kidney, and heart did not show an increase in lipoprotein receptors after hCG injection. An examination of the equilibrium dissociation constants for 125I-LDL and 125I-HDL binding after hCG administration revealed that the increase in binding activity was due to an increase in the number of binding sites rather than to a change in the binding affinity. In conclusion, rat corpus luteum possesses specific receptors for both LDL and HDL and these receptors are regulated by gonadotropins.