RESUMEN
Freshly isolated rat hepatocytes bind the solely apolipoprotein B-containing human low density lipoprotein (LDL) with a high-affinity component. After 1 h of incubation less than 30% of the cell-associated human LDL is internalized and no evidence for any subsequent high-affinity degradation was obtained. Scatchard analysis of the binding data for human 125I-labeled LDL indicates that the high-affinity receptor for human LDL on rat hepatocytes possesses a Kd of 2.6 x 10(-8)M, while the binding is dependent on the extracellular Ca2+ concentration. Competition experiments indicate that both the apolipoprotein B-containing lipoproteins (human LDL and rat LDL) as well as the apolipoprotein E-containing lipoproteins (human HDL and rat HDL) do compete for the same surface receptor. It is concluded that hepatocytes freshly isolated from untreated rats do contain, in addition to the earlier described rat lipoprotein receptor which does not interact with human apolipoprotein B-containing LDL, a high-affinity receptor which interacts both with solely apolipoprotein B-containing human LDL and apolipoprotein E-containing lipoproteins.
Asunto(s)
Lipoproteínas LDL/metabolismo , Hígado/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Unión Competitiva , Membrana Celular/metabolismo , Humanos , Cinética , Lipoproteínas HDL/aislamiento & purificación , Lipoproteínas LDL/aislamiento & purificación , Masculino , Ratas , Ratas Endogámicas , Receptores de LDLRESUMEN
Chemical modification of lysine or arginine residues of apolipoprotein B-100 in human low-density lipoprotein (LDL) with respectively reductive methylation (Me-LDL) or cyclohexanedione treatment (CHD-LDL) was applied to determine the role of these amino acids in LDL recognition by the various liver cell types. The cell association of native human LDL, Me-LDL and CHD-LDL to parenchymal and non-parenchymal cells was determined in vivo by isolating the various cell types 30 min after intravenous injection of the lipoproteins. In order to prevent degradation or release of cell-bound apolipoproteins during cell dissociation and purification, a low-temperature (8 degrees C) liver perfusion and cell isolation procedure was performed. It was found that reductive methylation of LDL inhibits the association of LDL to both parenchymal and non-parenchymal cells, indicating that lysine residues are important for recognition of LDL by both these cell types. In contrast, cyclohexanedione treatment of LDL did not influence the cell association of LDL to non-parenchymal cells. 17 alpha-Ethinyl estradiol treatment selectively increases the cell association of LDL by parenchymal cells (16-fold), leaving the non-parenchymal cell association uninfluenced. The increased cell-association of LDL to parenchymal cells is almost completely blocked by cyclohexanedione treatment of LDL (by 81%) or by methylation of LDL (by 97%). These data indicate that the arginine residues in LDL are not important for the recognition of LDL by non-parenchymal cells, whereas for the cell association of LDL to the estrogen-stimulated binding site on parenchymal cells both arginine and lysine residues are essential. The in vivo cell association of CHD-LDL or native LDL to non-parenchymal cells was lowered to the level of Me-LDL by ethyl oleate treatment of the rats, while no effect of ethyl oleate on parenchymal cells was noticed. These data suggest that the specific site for LDL on non-parenchymal cells, which need lysine residues on LDL for recognition, can be down-regulated by ethyl oleate treatment. The LDL, internalized by non-parenchymal cells, is effectively degraded. This degradation occurs at least partly in the lysosomes. It is suggested that the unique recognition site for LDL on non-parenchymal cells may be quantitatively important for serum LDL catabolism.
Asunto(s)
Etinilestradiol/farmacología , Lipoproteínas LDL/metabolismo , Hígado/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Ciclohexanonas/farmacología , Masculino , Metilación , Ácidos Oléicos/farmacología , Perfusión , Ratas , Ratas Endogámicas , Receptores de LDL , Distribución TisularRESUMEN
The cellular localization in the liver of the receptor-dependent and -independent uptake of human low density lipoprotein (LDL) in normal and 17 alpha-ethinyl estradiol-treated rats was investigated by the simultaneous in vivo injection of human 131I-LDL and human reductive methylated 125I-LDL. The cells were subsequently isolated by a low temperature method. In untreated rats, after 30 min of in vivo circulation of human LDL, 57% of the receptor-dependent liver-association of human LDL occurs in non-parenchymal cells and 43% in parenchymal cells. Estradiol treatment of rats for 3 days selectively increases the receptor-dependent cell-association of human LDL with hepatocytes (17-fold), while the receptor-dependent cell-association with non-parenchymal cells is not affected.
Asunto(s)
Etinilestradiol/farmacología , Lipoproteínas LDL/metabolismo , Hígado/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Humanos , Lipoproteínas LDL/sangre , Hígado/citología , Masculino , Ratas , Ratas Endogámicas , Receptores de Superficie Celular/efectos de los fármacos , Receptores de LDLRESUMEN
The direct correlation between lipoprotein (a) (Lp(a)) concentrations and atherosclerosis stimulated us to investigate the in vivo interaction of Lp(a) with the liver and the various liver cell types. In untreated rats the serum decay of Lp(a) is comparable to that of LDL. By estrogen treatment the interaction of LDL with parenchymal liver cells is increased 17-fold whereas only a 2-fold effect on Lp(a) is found. The decay of Lp(a) in estrogen-treated rats is slower than for LDL. The data indicate that Lp(a) in vivo shows a less efficient interaction than LDL with the estrogen-induced apo-B,E receptor on parenchymal liver cells. It is suggested that the inability of Lp(a) to interact efficiently with the LDL removal system of the liver might be related to its atherogenic action.
Asunto(s)
Lipoproteínas/metabolismo , Hígado/metabolismo , Receptores de Lipoproteína , Animales , Endotelio/metabolismo , Etinilestradiol/farmacología , Humanos , Cinética , Macrófagos del Hígado/metabolismo , Lipoproteína(a) , Lipoproteínas/sangre , Lipoproteínas LDL/sangre , Hígado/efectos de los fármacos , Masculino , Metilación , Ratas , Ratas Endogámicas , Receptores de Superficie Celular/efectos de los fármacos , Receptores de Superficie Celular/metabolismo , Receptores de LDL/efectos de los fármacos , Receptores de LDL/metabolismoRESUMEN
The effect of different proportions of casein in semipurified diets on the concentration of serum cholesterol and the lipoprotein composition was studied in rabbits. Low-casein diets (10% w/w) resulted in serum cholesterol levels and growth rates that were lower than high-casein diets (40%). An intermediate proportion of casein (20%) produced intermediate concentrations of serum cholesterol, but only minor differences in food intake and weight gain, compared with the high-casein group. In the animals with the highest values of total serum cholesterol (the 40% casein group), most of the serum cholesterol was transported in the very low density lipoproteins, whereas with moderate hypercholesterolemia (the 20% casein group), the low density lipoproteins were the main carriers of cholesterol. Elevation in lipoprotein cholesterol was associated in all groups with an increased ratio of cholesterol to protein, suggesting the formation of particles relatively rich in cholesterol. When the rabbits on the diet containing 10% casein were subsequently transferred to the 40% casein diet, a steep increase in the level of serum cholesterol occurred. Conversely, switching the rabbits on the 40% casein diet to the 10% casein diet resulted in a decrease in the level of serum cholesterol.
Asunto(s)
Caseínas/administración & dosificación , Colesterol/sangre , Proteínas en la Dieta/administración & dosificación , Lipoproteínas/sangre , Animales , Proteínas Sanguíneas/metabolismo , Peso Corporal/efectos de los fármacos , HDL-Colesterol , LDL-Colesterol , VLDL-Colesterol , Relación Dosis-Respuesta a Droga , Hipercolesterolemia/sangre , Lipoproteínas HDL/sangre , Lipoproteínas LDL/sangre , Lipoproteínas VLDL/sangre , Masculino , ConejosAsunto(s)
Asialoglicoproteínas , Lipoproteínas/metabolismo , Hígado/citología , Receptores de Superficie Celular/fisiología , Animales , Autorradiografía , Radioisótopos de Carbono , Separación Celular/métodos , Endotelio/ultraestructura , Fetuínas , Macrófagos del Hígado/ultraestructura , Lipoproteínas LDL/administración & dosificación , Hígado/ultraestructura , Ratas , Receptores de Superficie Celular/aislamiento & purificación , Receptores de Lipoproteína , Temperatura , alfa-Fetoproteínas/metabolismoRESUMEN
In order to assess the relative importance of the receptor for low-density lipoprotein (LDL) (apo-B,E receptor) in the various liver cell types for the catabolism of lipoproteins in vivo, human LDL was labelled with [14C]sucrose. Up to 4.5h after intravenous injection, [14C]sucrose becomes associated with liver almost linearly with time. During this time the liver is responsible for 70-80% of the removal of LDL from blood. A comparison of the uptake of [14C]sucrose-labelled LDL and reductive-methylated [14C]sucrose-labelled LDL ([14C]sucrose-labelled Me-LDL) by the liver shows that methylation leads to a 65% decrease of the LDL uptake. This indicated that 65% of the LDL uptake by liver is mediated by a specific apo-B,E receptor. Parenchymal and non-parenchymal liver cells were isolated at various times after intravenous injection of [14C]sucrose-labelled LDL and [14C]sucrose-labelled Me-LDL. Non-parenchymal liver cells accumulate at least 60 times as much [14C]sucrose-labelled LDL than do parenchymal cells accumulate at least 60 times as much [14C]sucrose-labelled LDL than do parenchymal cells when expressed per mg of cell protein. This factor is independent of the time after injection of LDL. Taking into account the relative protein contribution of the various liver cell types to the total liver, it can be calculated that non-parenchymal cells are responsible for 71% of the total liver uptake of [14C]sucrose-labelled LDL. A comparison of the cellular uptake of [14C]sucrose-labelled LDL and [14C]sucrose-labelled Me-LDL after 4.5h circulation indicates that 79% of the uptake of LDL by non-parenchymal cells is receptor-dependent. With parenchymal cells no significant difference in uptake between [14C]sucrose-labelled LDL and [14C]sucrose-labelled Me-LDL was found. A further separation of the nonparenchymal cells into Kupffer and endothelial cells by centrifugal elutriation shows that within the non-parenchymal-cell preparation solely the Kupffer cells are responsible for the receptor-dependent uptake of LDL. It is concluded that in rats the Kupffer cell is the main cell type responsible for the receptor-dependent catabolism of lipoproteins containing only apolipoprotein B.
Asunto(s)
Lipoproteínas LDL/metabolismo , Hígado/metabolismo , Sacarosa/metabolismo , Animales , Lipoproteínas LDL/sangre , Hígado/citología , Masculino , Metilación , Ratas , Ratas Endogámicas , Receptores de LDL/metabolismo , Sacarosa/sangre , Factores de Tiempo , Distribución TisularRESUMEN
Cholesteryl-ester-rich very-low-density lipoproteins (beta-VLDL) are considered to be atherogenic because in vitro they can provoke cholesterol accumulation in macrophages. The greatest population of macrophages resides inside the liver and in the present study the rat beta-VLDL uptake by the various rat liver cell types is determined in vivo and compared to the uptake of rat VLDL. beta-VLDL isolated from cholesterol-fed rats was iodinated and injected into the rat. After 10 min of circulation, 45% of the injected beta-VLDL was found in the liver. A low-temperature cell-isolation procedure shows that rat liver parenchymal cells form the major site for beta-VLDL uptake (96%) and, consequently, rat liver macrophages (nonparenchymal liver cells) do not perform a quantitatively significant role in the uptake of these lipoproteins. In vitro competition studies indicate that apolipoprotein (apo) E is the site recognised by liver parenchymal cells and even a 600-fold excess of apo-E-free human LDL was an ineffective competitor. Furthermore it can be demonstrated that induction of apo-B,E receptors on liver parenchymal cells by estrogen treatment does not result in a significant increased uptake of beta-VLDL. These data show that recognition of beta-VLDL is presumably exerted by the remnant receptor. Intracellular processing of both the apolipoproteins and phospholipids of beta-VLDL was followed by subcellular distribution studies. It appears that, within 45 min, 75% of the apolipoproteins are degraded and subsequently released from the liver. In contrast the phospholipids remain associated with the liver for a prolonged time and a specific transfer to the mitochondrial fraction is found. It can be concluded that liver parenchymal cells form in vivo the major site for beta-VLDL uptake and it appears that recognition of beta-VLDL is coupled to internalization and processing of both the apolipoproteins and phospholipids by a route which involves the lysosomes.
Asunto(s)
Lipoproteínas VLDL/metabolismo , Hígado/metabolismo , Animales , Apolipoproteínas/metabolismo , Separación Celular , Colesterol en la Dieta/administración & dosificación , Colesterol en la Dieta/metabolismo , Técnicas In Vitro , Hígado/citología , Lisosomas/metabolismo , Macrófagos/metabolismo , Masculino , Fosfolípidos/metabolismo , Ratas , Ratas Endogámicas , Receptores de LDL/metabolismo , Fracciones Subcelulares/metabolismo , TemperaturaRESUMEN
1. Modified lipoproteins have been implicated to play a significant role in the pathogenesis of atherosclerosis. In view of this we studied the fate and mechanism of uptake in vivo of acetylated human low-density lipoprotein (acetyl-LDL). Injected intravenously into rats, acetyl-LDL is rapidly cleared from the blood. At 10min after intravenous injection, 83% of the injected dose is recovered in liver. Separation of the liver into a parenchymal and non-parenchymal cell fraction indicates that the non-parenchymal cells contain a 30-50-fold higher amount of radioactivity per mg of cell protein than the parenchymal cells. 2. When incubated in vitro, freshly isolated non-parenchymal cells show a cell-association of acetyl-LDL that is 13-fold higher per mg of cell protein than with parenchymal cells, and the degradation of acetyl-LDL is 50-fold higher. The degradation of acetyl-LDL by both cell types is blocked by chloroquine (10-50mum) and NH(4)Cl (10mm), indicating that it occurs in the lysosomes. Competition experiments indicate the presence of a specific acetyl-LDL receptor and degradation pathway, which is different from that for native LDL. 3. Degradation of acetyl-LDL by non-parenchymal cells is completely blocked by trifluoperazine, penfluridol and chlorpromazine with a relative effectivity that corresponds to their effectivity as calmodulin inhibitors. The high-affinity degradation of human LDL is also blocked by trifluoperazine (100mum). The inhibition of the processing of acetyl-LDL occurs at a site after the binding-internalization process and before intralysosomal degradation. It is suggested that calmodulin, or a target with a similar sensitivity to calmodulin inhibitors, is involved in the transport of the endocytosed acetyl-LDL to or into the lysosomes. 4. It is concluded that the liver, and in particular non-parenchymal liver cells, are in vivo the major site for acetyl-LDL uptake. This efficient uptake and degradation mechanism for acetyl-LDL in the liver might form in vivo the major protection system against the potential pathogenic action of modified lipoproteins.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Calmodulina/metabolismo , Lipoproteínas LDL/metabolismo , Hígado/metabolismo , Cloruro de Amonio/farmacología , Animales , Antipsicóticos/farmacología , Calcio/farmacología , Cloroquina/farmacología , Humanos , Técnicas In Vitro , Hígado/citología , Hígado/efectos de los fármacos , Ratas , Distribución Tisular , Trifluoperazina/farmacologíaRESUMEN
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 low density lipoproteins (LDL) in varying quantities. Upon addition to LDL, Tris-Gal-Chol was immediately incorporated, and after intravenous injection into rats, the iodine-labeled apolipoprotein B radioactivity was readily associated with the liver. The incorporation of 5 or 13 micrograms of Tris-Gal-Chol into LDL (20 micrograms of protein) stimulates the parenchymal cell association of LDL 6- and 10-fold, respectively, at 10 min after injection. For non-parenchymal cells, the cell association is 60- and 70-fold stimulated, respectively. It can be calculated that non-parenchymal cells (mainly Kupffer cells) are for 80-90% responsible for the increased, galactose-mediated, interaction of Tris-Gal-Chol LDL with the liver. The increased interaction of LDL with the cells upon Tris-Gal-Chol incorporation is followed by degradation of the apolipoprotein B in the lysosomes. Incorporation of Tris-Gal-Chol into unilamellar liposomes (10 mol %) leads to an increased cell association of the enclosed [3H]inulin to parenchymal cells (1.4-fold) and non-parenchymal cells (11.8-fold). It is concluded that Tris-Gal-Chol incorporation into LDL leads to a markedly increased catabolism of LDL by the liver which might be used for lowering serum LDL levels. The possibility of increasing the interaction of LDL or liposomes with specific liver cell types by Tris-Gal-Chol might also have an application for targeting drugs or other compounds of interest to these cells.