Effects of cholestyramine on receptor-mediated plasma clearance and tissue uptake of human low density lipoproteins in the rabbit

This study examines the effects of cholestyramine (2 g/day) on the plasma clearance and tissue uptake of human low density lipoprotein (LDL) in rabbits. 1,2-Cyclohexanedione modification of human LDL abolishes its recognition by high affinity cell membrane receptors in vitro and delays its plasma clearance in comparison to native LDL. Consequently, the difference between the fractional rates of catabolism of simultaneously injected native and cyclohexanedione-treated LDL is an index of in vivo receptor-mediated clearance of the lipoprotein. When human 125I-LDL and 131I-cyclohexanedione-treated LDL were injected into rabbits, 44% of the lipoprotein was cleared from the plasma by the receptor mechanism. Various tissues were removed from the animals at the end of the turnover study and their relative uptakes of 125I native and 131I-cyclohexanedione-treated LDL were measured. All exhibited receptor activity to some extent, incorporating more native than cyclohexanedione-modified LDL. The greatest receptor activity per g of tissue was found in lymph nodes, spleen, and liver and, in terms of whole organ uptake, the liver played a major role in LDL catabolism. Treatment of the rabbits with cholestyramine lowered the circulating LDL cholesterol level by promoting its clearance (120%, p < 0.001) via the receptor pathway. This was associated with a virtual doubling of receptor-mediated incorporation of the lipoprotein into the liver. These results suggest that the drain which cholestyramine induces in the hepatic cholesterol pool promotes LDL receptor activity in this organ and thereby lowers the level of circulating LDL.     

Uptake of LDL in parenchymal and non-parenchymal rabbit liver cells in vivo. LDL uptake is increased in endothelial cells in cholesterol-fed rabbits.

1. Hepatic uptake of low-density lipoprotein (LDL) in parenchymal cells and non-parenchymal cells was studied in control-fed and cholesterol-fed rabbits after intravenous injection of radioiodinated native LDL (125I-TC-LDL) and methylated LDL (131I-TC-MetLDL). 2. LDL was taken up by rabbit liver parenchymal cells, as well as by endothelial and Kupffer cells. Parenchymal cells, however, were responsible for 92% of the hepatic LDL uptake. 3. Of LDL in the hepatocytes, 89% was taken up via the B,E receptor, whereas 16% and 32% of the uptake of LDL in liver endothelial cells and Kupffer cells, respectively, was B,E receptor-dependent. 4. Cholesterol feeding markedly reduced B,E receptor-mediated uptake of LDL in parenchymal liver cells and in Kupffer cells, to 19% and 29% of controls, respectively. Total uptake of LDL in liver endothelial cells was increased about 2-fold. This increased uptake is probably mediated via the scavenger receptor. The B,E receptor-independent association of LDL with parenchymal cells was not affected by the cholesterol feeding. 5. It is concluded that the B,E receptor is located in parenchymal as well as in the non-parenchymal rabbit liver cells, and that this receptor is down-regulated by cholesterol feeding. Parenchymal cells are the main site of hepatic uptake of LDL, both under normal conditions and when the number of B,E receptors is down-regulated by cholesterol feeding. In addition, LDL is taken up by B,E receptor-independent mechanism(s) in rabbit liver parenchymal, endothelial and Kupffer cells. The non-parenchymal liver cells may play a quantitatively important role when the concentration of circulating LDL is maintained at a high level in plasma, being responsible for 26% of hepatic uptake of LDL in cholesterol-fed rabbits as compared with 8% in control-fed rabbits. The proportion of hepatic LDL uptake in endothelial cells was greater than 5-fold higher in the diet-induced hypercholesterolaemic rabbits than in controls.     

Receptor-independent low-density lipoprotein catabolism: Evaluation of 2-hydroxyacetaldehyde-treated lipoprotein as a probe for its measurement

This study examines the protein modification procedures available for inhibiting receptor recognition of low-density lipoprotein (LDL). Glycosylation with glucose, idose or ribose blocks the interaction of the lipoprotein with the high-affinity LDL receptor on cultured fibroblast membranes and delays its clearance from the plasma of rabbits. However, the prolonged incubation required in the process also changes the metabolic properties of the lipoprotein. An alternative approach using 2-hydroxyacetaldehyde-treated LDL completely blocks receptor recognition. This modified tracer has the same metabolic properties as the reductively methylated lipoprotein in rabbits and appears to be a suitable probe for the measurement of the receptor-independent LDL catabolic pathway in humans.     

Receptor-independent low-density lipoprotein catabolism. Evaluation of 2-hydroxyacetaldehyde-treated lipoprotein as a probe for its measurement

This study examines the protein modification procedures available for inhibiting receptor recognition of low-density lipoprotein (LDL). Glycosylation with glucose, idose or ribose blocks the interaction of the lipoprotein with the high-affinity LDL receptor on cultured fibroblast membranes and delays its clearance from the plasma of rabbits. However, the prolonged incubation required in the process also changes the metabolic properties of the lipoprotein. An alternative approach using 2-hydroxyacetaldehyde-treated LDL completely blocks receptor recognition. This modified tracer has the same metabolic properties as the reductively methylated lipoprotein in rabbits and appears to be a suitable probe for the measurement of the receptor-independent LDL catabolic pathway in humans.     

Role of a calf thymus preparation in the degradation of native and reductively methylated low density lipoprotein.

1. The clearance of low density lipoprotein (LDL) is mediated by a specific LDL receptor pathway and by an alternative metabolic pathway that is responsible for the receptor-independent LDL catabolism. 2. This alternative catabolism can be studied in vivo using a preparation of chemically modified LDL that are reductively methylated. 3. Recently we showed that a calf thymus protein extract affects the cholesterol metabolism via activation of LDL catabolism. 4. The aim of this study was to investigate whether in vivo the specific LDL receptor pathway and the independent LDL receptor pathway are affected by thymus treatment. 5. The results obtained injecting in rats native and chemically modified 125I-LDL to probe the receptor independent pathway, show that the thymus gland decreases serum cholesterol by activation of the specific LDL receptor pathway. 6. This effect is mainly evident in liver and kidney that represent organs in which the specific LDL receptors are widely present.     

Partial ileal bypass reduces the production rate of low density lipoproteins in Watanabe heritable hyperlipidemic rabbits

Partial ileal bypass surgery in homozygous Watanabe heritable hyperlipidemic (WHHL) rabbits resulted in a decrease of low density lipoproteins (LDL)-cholesterol from 14.2 +/- 2.4 to 7.0 +/- 1.2 mmol/l. To investigate the effect of partial ileal bypass on receptor-mediated and receptor-independent LDL catabolism, turnover studies were performed of radiolabeled native LDL and chemically modified LDL (methyl-LDL) in WHHL rabbits after partial ileal bypass, in WHHL control rabbits, and in New Zealand White ("normal") rabbits. The plasma LDL pool in WHHL control rabbits was increased 10-fold. The receptor-mediated LDL clearance was essentially zero in WHHL rabbits, both in controls and after ileal bypass surgery; the fractional catabolic rates for total LDL were equal in both WHHL groups and were also similar to that for methyl-LDL in the normal rabbits. Seventy percent of the total LDL clearance in the normal rabbits occurred via the LDL receptor pathway. In the animals with a partial ileal bypass, the plasma LDL-protein pool was appreciably lower than in WHHL controls (41.6 +/- 5.7 vs 73.4 +/- 9.9 mg/kg, P less than 0.02). The absolute catabolic rate was almost 50% lower in the PIB group (21.4 +/- 2.0 vs 40.0 +/- 7.5 mg X kg-1 X day-1, P less than 0.02). These results indicate that the decrease of LDL after partial ileal bypass surgery in WHHL rabbits is the result of a reduced production rate of LDL.     

Role of receptor-independent low density lipoprotein transport in the maintenance of tissue cholesterol balance in the normal and WHHL rabbit

These studies were undertaken to determine the role of receptor-independent low density lipoprotein (LDL) transport in cholesterol balance across individual tissues and the whole animal. Homologous LDL, which measures total LDL transport, and methylated heterologous LDL, which measures receptor-independent LDL uptake, were cleared from the plasma at very different rates in the NZ control rabbit (3,900 and 1,010 microliter/hr per kg, respectively) whereas in the WHHL rabbit both preparations were cleared at essentially the same rate (approximately 1,070 microliter/hr per kg). Receptor-independent LDL clearance was detected in all tissues of the NZ control rabbit and these varied from 32 (spleen) to less than 0.5 (skeletal muscle) microliter/hr per g. In contrast, receptor-dependent LDL uptake was found in only about half of these same organs. In the WHHL rabbit, the rates of receptor-independent LDL transport were the same as in the NZ control rabbit, but no receptor-dependent uptake was detected. Using these clearance values it was calculated that in the control rabbit nearly 70% of LDL-cholesterol was removed from the plasma by the liver and 89% of this was receptor-mediated. With loss of receptor activity, however, the burden of LDL degradation was shifted away from the liver so that approximately 70% of LDL-cholesterol uptake took place in the extra-hepatic tissues of the WHHL rabbit. Thus, in the normal animal, the primary function of receptor-dependent LDL transport is to promote the rapid uptake and disposal of plasma LDL by the liver. In the absence of such receptor activity, cholesterol balance across most individual organs and the whole animal remains essentially normal and is mediated by the receptor-independent process. Because of the much lower absolute clearance rates manifested by this transport mechanism, however, substantial and predictable elevations in the circulating plasma LDL-cholesterol levels are required to maintain this balance.     

In vivo clearance of low density lipoprotein in pigeons occurs by a receptor-like mechanism that is not down-regulated by cholesterol feeding

The contribution of receptor-dependent and receptor-independent mechanisms for low density lipoprotein (LDL) clearance in vivo was determined in White Carneau and Show Racer pigeons fed either cholesterol free or cholesterol containing diets. The methylation of pigeon LDL resulted in the inhibition of recognition by the LDL receptor which allowed its use as a tracer of receptor-independent clearance. The fractional catabolic rate (FCR) of radiolabeled LDL in 20 control pigeons (means +/- S.E., 0.277 +/- 0.013 pools/h) was approximately seven times faster than for methylated LDL indicating that 86% of the total LDL clearance occurred by a receptor-mediated process. Total LDL clearance was reduced by 27% (FCR = 0.202 +/- 0.012 pools/h) in 14 cholesterol-fed pigeons, but receptor-mediated mechanisms were still responsible for 80% of the total LDL clearance. LDL uptake by individual tissues was measured using the residualizing label 125I-tyramine cellobiose. The liver was the primary site of LDL clearance in both control and cholesterol-fed birds. LDL receptors were active in every tissue examined and accounted for over 85% of the LDL clearance in the liver and over 90% in the adrenal gland. Consistent with the whole body LDL clearance findings, cholesterol-feeding did not significantly reduce receptor-mediated clearance of 125I-tyramine cellobiose-LDL by the liver or any of the other tissues. Hepatic sterol synthesis, however, was reduced by greater than 90% in cholesterol-fed animals. These data are consistent with the conclusion that LDL clearance in vivo in pigeons is mediated primarily by an LDL receptor-like mechanism that shows little down-regulation with hypercholesterolemia even though cholesterol synthesis is efficiently down-regulated.     

Tissue sites of degradation of native and reductively methylated [14C]sucrose-labeled low density lipoprotein in rats. Contribution of receptor-dependent and receptor-independent pathways

Low density lipoprotein (LDL) is catabolized by both receptor-dependent and receptor-independent pathways; methylated LDL (MeLDL) is catabolized only by receptor-independent mechanisms. Rats were injected with either LDL or MeLDL labeled with [14C]sucrose and the tissue sites of degradation were determined 24 h later. On degradation, the 14C-labeled ligand remains trapped intracellularly as a cumulative measure of degradation. The fractional catabolic rate (FCR) of [14C]sucrose-MeLDL was lower than that of [14C]sucrose-LDL (0.056 +/- 0.015 versus 0.118 +/- 0.025 h-1, p less than 0.01). Liver was the predominant site of catabolism of both LDL and MeLDL; more than 85% of catabolism was attributable to parenchymal cells in both cases. The fraction of the plasma LDL pool "cleared" per tissue weight per unit of time was determined for individual tissues. The differences in these rates for LDL and MeLDL are an approximation of receptor-mediated uptake of LDL. According to this method, 67.4% of hepatic uptake was attributable to receptors, as was 69.5% of adrenal, 65.4% of ovarian, 52.4% of intestinal, and 44.2% of renal uptake. In other studies, rats were continuously infused with LDL to down-regulate and saturate receptor prior to injection of labeled LDL or MeLDL. Rats infused with LDL exhibited a lower FCR for [14C]sucrose-LDL compared to controls (0.077 versus 0.120 h-1); the FCR for sucrose-MeLDL was unchanged by LDL infusion. The fractional degradation rate of [14C]sucrose-LDL by individual tissues was lowered by LDL infusion in liver, adrenal, ovary, and intestine (41.4, 57.3, 23.1, and 32.4% lower than controls, respectively). The determination of receptor dependency by this independent approach supports the conclusions reached using [14C]sucrose-LDL and [14C]sucrose-MeLDL in normolipemic animals.     

Regulation of guinea pig plasma low density lipoprotein kinetics by dietary fat saturation.

Dietary fat saturation has been shown to affect hepatic apoB/E receptor expression and to modify low density lipoprotein (LDL) composition and density in guinea pigs. The current studies were designed to investigate the independent and interactive effects of dietary fat saturation alterations in apoB/E receptor expression and LDL composition on in vivo LDL turnover kinetics, both receptor-mediated and receptor-independent. Guinea pigs were fed semi-purified diets containing 15% fat, either polyunsaturated corn oil (CO), monounsaturated olive oil (OL), or saturated lard, and injected with radioiodinated LDL isolated from animals fed the homologous diet. Blood samples were obtained over 33 h to determine apoLDL fractional catabolic rates (FCR) and flux rates. Compared to animals fed OL- or lard-based diets, intake of the CO-based diet resulted in a 50% decrease in LDL apoB pool size associated with a twofold increase in receptor-mediated FCR (P less than 0.001) and a 28% decrease in flux rate (P less than 0.05). Maximal LDL binding capacity of hepatic apoB/E receptors, determined in vitro, was twofold higher for animals fed the CO-based diet compared to guinea pigs fed the OL- and lard-based diets (P less than 0.01). There was a significant correlation between hepatic apoB/E receptor number and in vivo receptor-mediated LDL FCR (r = 0.987). Significant differences in LDL turnover were related to the source of LDL. When injected into animals fed a nonpurified commercial diet, the smaller, cholesteryl ester-depleted LDL isolated from animals fed the CO-based diet had a twofold higher FCR compared to larger LDLs from guinea pigs fed the OL- and lard-based diets, which had similar turnover rates. When LDL from animals fed the commercial diet was radiolabeled and injected into animals fed the three types of dietary fat, significant differences in LDL turnover were observed in the order CO greater than lard greater than OL, suggesting that intravascular processing and tissue uptake of the smaller LDL from animals fed the commercial diet varies depending on the dietary fat saturation fed to the recipient animals. These studies demonstrate that guinea pigs fed polyunsaturated fat diets lower plasma LDL levels in part by an increase in apoB/E receptor-mediated fractional LDL turnover and a decrease in apoLDL flux. In addition, fat saturation alters LDL composition and size which independently affect LDL turnover rates in vivo.     

Stimulation of receptor-dependent and receptor-independent pathways of low-density lipoprotein degradation in arterial smooth muscle cells by platelet-derived growth factor

Platelet-derived growth factor (PDGF), a powerful mitogen released by platelets, promoted the degradation of low-density lipoprotein (LDL) by cultured primate arterial smooth muscle cells and human skin fibroblasts by stimulating both receptor-mediated and LDL-receptor-independent uptake of LDL. Stimulation of LDL-receptor-independent LDL uptake and degradation by PDGF was demonstrated in three ways. First, the small amount of LDL that was degraded by LDL-receptor-negative skin fibroblasts was stimulated by PDGF. Second, PDGF led to increased degradation of LDL that had been reductively methylated to prevent its binding to LDL receptors. Third, 125I-labeled LDL degradation was stimulated by PDGF in the presence of high concentrations of unlabeled LDL, i.e., conditions under which the contribution of the LDL receptor to cellular uptake and degradation is reduced. These observations suggest that mitogens, as typified by PDGF, can facilitate the cellular delivery of LDL cholesterol by both LDL-receptor-mediated and non-LDL-receptor-mediated mechanisms to provide exogenous cholesterol for use during cell replication.     

Uptake and degradation of low density lipoproteins in atherosclerotic rabbit aorta: role of local LDL modification

The uptake of native and modified low density lipoprotein (LDL) in foam cells in atherosclerotic tissue was studied in an in vitro perfusion system for rabbit aorta. Experimental atherosclerosis was induced in rabbits by a combination of cholesterol feeding and mechanical injury. The aorta was perfused in an incubation chamber. A trace-label, radioiodinated tyramine-cellobiose, was used to study cellular uptake of lipoproteins. After perfusion, the tissue was digested and cells were isolated by centrifugation in a density gradient. About 40 times more LDL per cell was accumulated in the foam cell fraction than in the smooth muscle cell fraction. When the cellular uptake of LDL and acetylated LDL (AcLDL) was compared, about 4 times more AcLDL than LDL was taken up by the foam cells, suggesting that the scavenger receptor is expressed in these cells. In a competition experiment, the uptake of LDL into foam cells was reduced by 70% when a tenfold excess of AcLDL was added. This experiment suggests that native LDL is taken up by the same mechanism as AcLDL. The accumulation of radiolabeled LDL in plaques and in foam cells was reduced by 30-55% by adding vitamin E (0.1 mg/ml) to the system. These studies show an uptake of LDL by foam cells in the atherosclerotic tissue. Furthermore, these cells seem to express the scavenger receptor. The competition experiment would suggest that native LDL is taken up by the scavenger receptor. The observation that an antioxidant, vitamin E, may decrease this uptake suggests that oxidative modification of LDL is of importance for this process.     

Minimally oxidized LDL inhibits macrophage selective cholesteryl ester uptake and native LDL-induced foam cell formation

Scavenger receptor-mediated uptake of oxidized LDL (oxLDL) is thought to be the major mechanism of foam cell generation in atherosclerotic lesions. Recent data has indicated that native LDL is also capable of contributing to foam cell formation via low-affinity receptor-independent LDL particle pinocytosis and selective cholesteryl ester (CE) uptake. In the current investigation, Cu²⁺-induced LDL oxidation was found to inhibit macrophage selective CE uptake. Impairment of selective CE uptake was significant with LDL oxidized for as little as 30 min and correlated with oxidative fragmentation of apoB. In contrast, LDL aggregation, LDL CE oxidation, and the enhancement of scavenger receptor-mediated LDL particle uptake required at least 3 h of oxidation. Selective CE uptake did not require expression of the LDL receptor (LDL-R) and was inhibited similarly by LDL oxidation in LDL-R^−/− versus WT macrophages. Inhibition of selective uptake was also observed when cells were pretreated or cotreated with minimally oxidized LDL, indicating a direct inhibitory effect of this oxLDL on macrophages. Consistent with the effect on LDL CE uptake, minimal LDL oxidation almost completely prevented LDL-induced foam cell formation. These data demonstrate a novel inhibitory effect of mildly oxidized LDL that may reduce foam cell formation in atherosclerosis.     

Visualization of the binding, endocytosis, and transcytosis of low- density lipoprotein in the arterial endothelium in situ

We investigated the interaction and transport of low-density lipoprotein (LDL) through the arterial endothelium in rat aorta and coronary artery, by perfusing in situ native, untagged human, and rat LDL. The latter was rendered electron-opaque after it interacted with the endothelial cell and was subsequently fixed within tissue. We achieved LDL electron-opacity by an improved fixation procedure using 3,3'-diaminobenzidine, and mordanting with tannic acid. The unequivocal identification of LDL was implemented by reacting immunocytochemically the perfused LDL with anti LDL-horseradish peroxidase conjugate. Results indicate that LDL is taken up and internalized through two parallel compartmented routes. (a) A relatively small amount of LDL is taken up by endocytosis via: (i) a receptor-mediated process (adsorptive endocytosis) that involved coated pits/vesicles, and endosomes, and, probably, (ii) a receptor-independent process (fluid endocytosis) carried out by a fraction of plasmalemmal vesicles. Both mechanisms bringing LDL to lysosomes supply cholesterol to the endothelial cell itself. (b) Most circulating LDL is transported across the endothelial cell by transcytosis via plasmalemmal vesicles which deliver LDL to the other cells of the vessel wall. Endocytosis is not enhanced by increasing LDL concentration, but the receptor-mediated internalization decreases at low temperature. Transcytosis is less modified by low temperature but is remarkably augmented at high concentration of LDL. While the endocytosis of homologous (rat) LDL is markedly more pronounced than that of heterologous (human) LDL, both types of LDL are similarly transported by transcytosis. These results indicate that the arterial endothelium possesses a dual mechanism for handling circulating LDL: by a high affinity process, endocytosis secures the endothelial cells' need for cholesterol; by a low-affinity nonsaturable uptake process, transcytosis supplies cholesterol to the other cells of the vascular wall, and can monitor an excessive accumulation of plasma LDL. Since in most of our experiments we used LDL concentrations above those found in normal rats, we presume that at low LDL concentrations saturable high-affinity uptake would be enhanced in relation to nonsaturable pathways.     

Turnover and tissue sites of degradation of glucosylated low density lipoprotein in normal and immunized rabbits

Immunological mechanisms have been implicated in the atherogenic process since immunoglobulins are frequently found in the atherosclerotic aorta. We have previously shown that modifications of homologous low density lipoproteins (LDL) make it immunogenic. In particular we have demonstrated that immunization with homologous nonenzymatically glucosylated LDL (glcLDL) results in the generation of antibodies specific to the derivatized lysine residue, and that such antibodies do not react with native LDL epitopes. In the present study we immunized rabbits with reductively glucosylated rabbit LDL and then determined the effects of the circulating antibodies on the rates of plasma clearance and on the sites of degradation of LDL in which varying degrees of glucosylation had been achieved. In normal chow-fed animals, the plasma clearance of glcLDL was retarded in proportion to the extent of lysine derivatization. In contrast, in immunized animals the clearance of glcLDL was greatly accelerated. When 10% or more of lysine residues were derivatized, clearance of glcLDL was accelerated 50- to 100-fold. Even when only 5% of lysines were derivatized, plasma clearance was accelerated 2- to 3-fold. Cholesterol feeding inhibited LDL clearance from plasma and decreased LDL uptake of LDL receptor-rich tissues. In a similar manner, glucosylation of LDL inhibited its ability to bind to the LDL receptor and redirected sites of LDL degradation away from LDL receptor-rich tissues. Thus degradation of glcLDL by liver and adrenal was markedly diminished. The presence of antibodies to glcLDL also redirected sites of degradation of the modified LDL, primarily to the reticuloendothelial cells of the liver. There was no evidence for specific targeting of glcLDL-immunoglobulin complexes to the aorta; instead they were targeted to the liver. These data suggest that the presence of humoral antibodies to modified LDL acts to rapidly remove such LDL from plasma and specifically targets such complexes to reticuloendothelial cells, primarily in the liver. In this manner such antibodies may serve a useful purpose.     

The uptake of chylomicron remnants and very low density lipoprotein remnants by the perfused rat liver

The regulation of the hepatic uptake of chylomicron remnants and very-low-density lipoprotein (VLDL) remnants was studied in the rat using a nonrecirculating liver perfusion system. The hepatic removal of remnant lipoproteins was shown to be by receptor-mediated processes since the concentration-dependent uptake was saturable and reductive methylation of the particles reduced the uptake of each lipoprotein by two-thirds. Treatment of liver donor rats with 17 alpha-ethinyl estradiol resulted in a 2-fold increase in the hepatic uptake of VLDL remnants, while cholesterol feeding of liver donor rats caused complete suppression of the receptor-mediated uptake of VLDL remnants. Chylomicron remnant removal was unaffected by estradiol administration and only slightly diminished by cholesterol feeding. The results of competition studies also indicated that a specific chylomicron remnant receptor exists in the liver. Apoprotein E was shown to be required for the receptor-mediated uptake of both remnant lipoproteins. Chylomicron remnants which contained no apoprotein E and VLDL remnants which contained reductively methylated apoprotein E were removed by the liver to about one-third of the extent of native particles. Thus the hepatic uptake of remnant lipoproteins occurs by receptor-mediated processes and the specific removal of both particles is mediated by apoprotein E. In addition, the uptake of VLDL remnants is regulated by the same factors that control hepatic low-density lipoprotein removal, while chylomicron remnant removal is unaffected by these factors.     

Modulation of low density lipoprotein receptor activity by bile acids: differential effects of chenodeoxycholic and ursodeoxycholic acids in the hamster

Hamsters were fed chenodeoxycholic acid (CDC), ursodeoxycholic acid, (UDC), or no bile acid. [14C]Sucrose-labeled hamster low density lipoprotein (LDL) and methylated human LDL were infused intravenously to study LDL receptor-dependent and LDL receptor-independent organ uptake, respectively, of LDL. Biliary CDC increased during both CDC and UDC treatment. The UDC enrichment of bile after UDC feeding was relatively small. Bile acid synthesis was suppressed after both bile acid treatments. Under the condition of an acute bile fistula, the hamster LDL uptake increased in the liver, heart, and adrenals in the CDC-treated animals. During an intact enterohepatic circulation, the hepatic uptake of hamster LDL, which accounted for a major portion of the total uptake, was increased after UDC treatment. The hamster LDL uptake in the colon, which represented only a small fraction of the total uptake, increased after CDC treatment. When hamster LDL was infused at increasing concentrations, its uptake was significantly higher in the UDC-treated than in the control and CDC-treated animals. The methylated human LDL uptake showed no significant changes in the different treatment groups under either experimental condition. The study shows significantly different effects of CDC and UDC on LDL receptor activity. Since these differences are expressed in spite of a similar suppression of bile acid synthesis, UDC may directly influence LDL receptor activity.     

Receptor-dependent and receptor-independent degradation of low density lipoprotein in normal rabbits and in receptor-deficient mutant rabbits

Low density lipoprotein (LDL) catabolism was studied using WHHL rabbits, an inbred strain deficient in LDL receptor activity and, thus, an animal model for homozygous familial hypercholesterolemia. WHHL and normal rabbits were injected with [14C]sucrose-LDL and the tissue sites of LDL degradation were determined 24 h later. On degradation of [14C]sucrose-LDL, the [14C]sucrose ligand remains trapped within tissues as a cumulative measure of degradation. The fractional catabolic rate of [14C]sucrose-LDL in Watanabe heritable hyperlipidemic (WHHL) rabbits was reduced (0.024 +/- 0.010 versus 0.063 +/- 0.026 h-1) but, by virtue of the increased plasma pool, total LDL flux was increased (33.5 +/- 9.6 versus 10.6 +/- 4.4 mg of LDL protein/kg/day). Liver was the predominant site of catabolism in both WHHL and normal rabbits (52.7 +/- 6.9 and 56.6 +/- 6.2% of total degradation). About 90% of hepatic catabolism was attributable to parenchymal cells in both cases. Thus, Kupffer cells, a major component of the reticuloendothelial system, do not play a major role in LDL catabolism in WHHL rabbits. Despite receptor deficiency, the relative contribution of various tissues to overall LDL degradation was not greatly altered and the absolute rate of delivery of LDL to all tissues was increased with the exception of the adrenal. Thus, there was no evidence that the increased degradation occurred in any special subset of "scavenger" cells. Nevertheless, local scavenger cell uptake may be critically important, especially in atherogenesis. If it is assumed that receptor-independent degradation occurs at the same rate in the tissues of WHHL and normal rabbits and that catabolism in the absence of receptors is a linear function of concentration, then one can estimate the fraction of uptake in normal tissues mediated by receptors. The difference in the fraction of the plasma LDL pool cleared per unit of time in normal and WHHL rabbits would reflect the contribution of receptors to fractional clearance. By this calculation, receptor-mediated degradation in normal rabbits was 62% overall, 63% in liver, 92% in adrenal, and 83% in gut.     

Synthetic low-density lipoprotein, a novel biomimetic lipid supplement for serum-free tissue culture

Lipid supplementation in serum-free tissue culture employs solubilization techniques to permit the addition of lipids, but these systems are potentially cytotoxic and do not present lipid in a natural form. In this research a simplified preparation method for synthetic low-density lipoprotein (sLDL) has been developed that involves microfluidization of a solvent lipid solution in a simple aqueous solution. This produces material with size and zeta potential characteristics similar to those of native LDL. sLDL supplementation in tissue culture media provides cholesterol concentrations higher than those achieved by 10% serum supplementation and existing chemically defined lipid supplements. sLDL stimulates NS0 and U937 cellular proliferation in completely serum-free media, the former in a lipid concentration dependent manner that is also related to both the receptor peptide structure employed and its concentration on the particle. The greatest NS0 cellular proliferation was obtained at the highest cholesterol concentration tested (0.5 mg/mL), which was 10 times higher than the cholesterol concentration achieved by standard 10% serum supplementation. U937 cellular proliferation was influenced by variation of sLDL's fatty acid constituents with a natural mixture producing maximal effect. Cell uptake studies in NS0 with fluorescently labeled sLDL indicated that assimilation is reduced by competition from native LDL. The planktonic nature of NS0 cell growth meant that cell binding and uptake experiments were difficult to conduct because of cellular aggregation. However, sLDL-induced U937 proliferation is ablated by the presence of an anti-LDL receptor antibody. The results indicate that sLDL uptake is via the LDL receptor and that sLDL can function as a lipid supplement for serum-free media capable of supplementation to cholesterol concentrations up to 0.5 mg/mL. Cellular uptake studies also suggest that sLDL will be useful for the targeting and delivery of materials to cells. sLDL therefore represents a new and promising synthetic biomimetic alternative to native LDL with multiple applications.     

Immunoelectron microscopic visualization of the transcytosis of low density lipoproteins in perfused rat arteries

A postembedding labeling technique was employed to visualize human native low density lipoproteins (LDL) during transcytosis in rat arterial endothelium. For this purpose human LDL was perfused through rat vasculature before fixation and processing for immunoelectron microscopy. The LDL particles were located on sections by anti-human apolipoprotein B-100 (LDL) antibodies and secondary antibodies or protein-A conjugated to 10-nm colloidal gold. LDL molecules were seen in plasmalemmal vesicles as well as in the subendothelial space. No colloidal gold was found in the intercellular junctions. Perfusion with reductively methylated LDL, which cannot bind to the LDL receptor, gave a similar labeling pattern, indicating that transcytosis of LDL via plasmalemmal vesicles is most likely receptor independent. Furthermore, the passage of LDL through intact vascular endothelium is a vesicular transport rather than an intercellular diffusion process.