Effects of cholestyramine on low density lipoprotein binding sites on liver membranes from rabbits with endogenous hypercholesterolemia induced by a wheat starch-casein diet

Rabbits fed a wheat starch-casein diet develop a marked hypercholesterolemia with a lipoprotein distribution similar to that of humans. Approximately 76% of the total cholesterol is carried in the low density lipoprotein (LDL) fraction (1.006 less than d less than 1.063 g/ml). Inclusion of 1% cholestyramine in the diet prevents the increase in plasma cholesterol. The cholestyramine effect is mediated through an increased fractional catabolic rate of 125I-LDL. In order to determine the potential role of hepatic LDL receptors in the removal of LDL from the plasma, binding of 125I-LDL and 125I-beta-VLDL (beta-migrating very low density lipoproteins) to hepatic membranes prepared from livers of rabbits fed the wheat starch-casein diet with or without cholestyramine supplementation was investigated. Membranes from livers of the cholestyramine-supplemented animals exhibit high levels of specific EDTA-sensitive binding of either of the 125I-labeled lipoproteins. Very little EDTA-sensitive binding occurs on liver membranes from wheat starch-casein-fed rabbits that have not been treated with cholestyramine. These results indicate that the hypercholesterolemia in rabbits associated with the wheat starch-casein diet is wholly or partially the result of a decreased number of specific hepatic LDL receptors and thus a decreased catabolism of plasma cholesterol. The response of the liver to the inclusion in the diet of the bile acid sequestrant, cholestyramine, is to maintain or increase the number of specific LDL binding sites, thus promoting catabolism of plasma cholesterol.     

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.     

Enhanced catabolism of low density lipoproteins in rat by lactosaminated Fab fragment. A new carrier of macromolecules to the liver

Proteins conjugated with lactose residues exhibit enhanced hepatic uptake mediated by the galactose receptor. In this study, we demonstrate that lactosaminated Fab fragments (lac-Fab) of IgG can induce hepatic catabolism of specific antigens, especially low density lipoproteins (LDL). lac-Fab and human LDL-lac-Fab complex exhibited specific uptake in isolated rat hepatocytes. In vivo in the rat, lactosamination enhanced plasma clearance of Fab fragments 2-fold and hepatic localization 20-fold. Fab fragments retained their affinity after lactosamination. Hepatic uptake of rat 125I-IgG complexed in vitro with anti-rat lac-Fab was increased almost 5-fold, compared to rat 125I-IgG alone. Injection of rats with anti-LDL lac-Fab induced plasma clearance and hepatic uptake of tracer amounts of previously injected human 125I-LDL, which decreased 50% 10 min after injection of lac-Fab, with 30% present in the liver. Asialofetuin completely inhibited these processes. After a bolus of 6 mg of human LDL, administration of anti-LDL lac-Fab reduced the serum cholesterol of rats to basal values within 2.5 h. These findings suggest that lactosaminated Fab fragments of specific IgGs are effective reagents for inducing hepatic uptake of macromolecules through the galactose receptor. lac-Fab specific for LDL may be an effective hypocholesterolemic agent in vivo.     

Receptor-mediated catabolism and tissue uptake of human low density lipoprotein in the cholesterol-fed, atherosclerotic rabbit

The LDL receptor pathway, which was delineated in cultured cells, is now known to operate in vivo. In this study we have measured the plasma clearances and tissue uptakes of native and chemically modified (1,2-cyclohexanedione-treated or reductively methylated) LDL in rabbits in order to determine the response of the pathway to a high-cholesterol diet. 1 week on the diet increased circulating LDL and suppressed its receptor-mediated plasma clearance and uptake into all tissues. The fractional catabolic rate of the lipoprotein via the receptor-independent route also fell. Continuation of the feeding program for 12 weeks accentuated these changes and virtually eliminated receptor uptake into all tissues so that the plasma decay curves of native and cyclohexanedione-treated LDL were superimposable. Lipoprotein assimilation by the aorta, however, did not follow this general trend. This tissue, after 12 weeks, was variably infiltrated by atheromatous deposits and the appearance of these lesions was associated with a substantial increase in the relative uptakes of both native and chemically modified (cyclohexanedione-treated and reductively methylated) LDL. We concluded (a) that expansion of tissue cholesterol pools virtually abolishes LDL receptor activity in rabbits; and (b) that LDL assimilation (both apparently receptor-mediated and receptor-independent) paradoxically increases at sites where the aorta is affected by atheromatous lesions.     

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.     

Residualizing and non-residualizing analogues of low-density lipoprotein as iodine-123 radiopharmaceuticals for imaging LDL catabolism

Low-density lipoprotein (LDL) labeled via direct iodination or via the radioiodinated residualizing moiety tyramine-cellobiose (TC) were compared in rabbits as potential ¹²³I radiopharmaceuticals for imaging sites of LDL catabolism. The tissue deposition of ¹³¹I-TC-LDL after 24 h as determined by dissection was in the major catabolic organs (liver, adrenals, spleen), and its plasma clearance was slower in rabbits with dietary hypercholesterolemia than in normals. ¹³¹I-LDL was unsuitable as a metabolic tracer due to redistribution of catabolites and/or loss of the label before protein degradation, which resulted in little accumulation of radioactivity in catabolic organs and high thyroid uptake. The plasma clearance half-time was similar (ca 22 h) for the two compounds in normal rabbits, but was increased to about 36 h for ¹³¹I-TC-LDL and decreased to approximately 9 h for ¹³¹I-LDL in hypercholesterolemic animals. The were similar with dynamic imaging of control and hypercholesterolemic rabbits using ¹²³I-labeled analogues. ¹²³I-TC-LDL rapidly localized in the liver, with low thyroid accumulation of radioactivity. The hepatic uptake of ¹²³I-LDL was about half that of ¹²³I-TC-LDL, and thyroid sequestration of radioactivity was significant for ¹²³I-LDL but not ¹²³I-TC-LDL. These data suggest that whereas the residualizing ¹²³I-TC-LDL has a pharmacokinetic profile representative of lipoprotein metabolism, the biodistribution of the activity from injected ¹²³I-LDL is complicated by processes other than protein degradation. The results are discussed with regard to nuclear medicine applications in evaluating lipoprotein catabolism in man.     

Mevinolin and cholestyramine inhibit the direct synthesis of low density lipoprotein apolipoprotein B in miniature pigs

Previous studies established that following simultaneous injection of 125I-labeled homologous very low density lipoproteins (VLDL) and 131I-labeled homologous low density lipoproteins (LDL) into miniature pigs, a large proportion of LDL apolipoprotein B (apoB) was synthesized directly, independent of VLDL or intermediate density lipoprotein (IDL) apoB catabolism. The possibility that cholestyramine alone (a bile acid sequestrant) or in combination with mevinolin (a cholesterol synthesis inhibitor) could regulate the direct LDL apoB synthetic pathway was investigated. 125I-labeled VLDL and 131I-labeled LDL were injected into miniature pigs (n = 8) during a control period and following 18 days of cholestyramine treatment (1.0 g kg-1d-1) or following 18 days of treatment with cholestyramine and mevinolin (1.2 mg kg-1d-1). ApoB in each lipoprotein fraction was selectively precipitated using isopropanol in order to calculate specific activity. In control experiments, LDL apoB specific activity curves reached their peak values well before crossing the VLDL or IDL apoB curves. However, cholestyramine treatment resulted in LDL apoB curves reaching maximal values much closer to the point of intersection with the VLDL or IDL curves. Kinetic analyses demonstrated that cholestyramine reduced total LDL apoB flux by 33%, which was due entirely to inhibition of the LDL apoB direct synthesis pathway since VLDL-derived apoB was unaffected. In addition, the LDL apoB pool size was reduced by 30% and the fractional catabolic rate of LDL apoB was increased by 16% with cholestyramine treatment. The combination of mevinolin and cholestyramine resulted in an even more marked inhibition of the direct LDL apoB synthesis pathway (by 90%), and in two animals this pathway was completely abolished. This inhibition was selective as VLDL-derived LDL apoB synthesis was not significantly different. LDL apoB pool size was reduced by 60% due primarily to the reduced synthesis as well as a 40% greater fractional removal rate. These results are consistent with the idea that cholestyramine and mevinolin increase LDL catabolism by inducing hepatic apoB, E receptors. We have now shown that the direct synthesis of LDL apoB is selectively inhibited by these two drugs.     

Lipoprotein [a] is cleared from the plasma primarily by the liver in a process mediated by apolipoprotein [a

The cellular and molecular mechanisms responsible for lipoprotein [a] (Lp[a]) catabolism are unknown. We examined the plasma clearance of Lp[a] and LDL in mice using lipoproteins isolated from human plasma coupled to radiolabeled tyramine cellobiose. Lipoproteins were injected into wild-type, LDL receptor-deficient (Ldlr-/-), and apolipoprotein E-deficient (Apoe-/-) mice. The fractional catabolic rate of LDL was greatly slowed in Ldlr-/- mice and greatly accelerated in Apoe-/- mice compared with wild-type mice. In contrast, the plasma clearance of Lp[a] in Ldlr-/- mice was similar to that in wild-type mice and was only slightly accelerated in Apoe-/- mice. Hepatic uptake of Lp[a] in wild-type mice was 34.6% of the injected dose over a 24 h period. The kidney accounted for only a small fraction of tissue uptake (1.3%). To test whether apolipoprotein [a] (apo[a]) mediates the clearance of Lp[a] from plasma, we coinjected excess apo[a] with labeled Lp[a]. Apo[a] acted as a potent inhibitor of Lp[a] plasma clearance. Asialofetuin, a ligand of the asialoglycoprotein receptor, did not inhibit Lp[a] clearance. In summary, the liver is the major organ accounting for the clearance of Lp[a] in mice, with the LDL receptor and apolipoprotein E having no major roles. Our studies indicate that apo[a] is the primary ligand that mediates Lp[a] uptake and plasma clearance.     

Regulation of hepatic receptor-dependent degradation of LDL by mevinolin in rabbits with hypercholesterolemia induced by a wheat starch-casein diet

Rabbits fed a wheat starch-casein diet develop a marked hypercholesterolemia and have a slower rate of removal of rabbit 125I-labeled low density lipoproteins (LDL) from plasma. Treating rabbits with mevinolin, a highly potent competitive inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, at a daily dose of 20 mg per animal prevents the increase in plasma and LDL cholesterol. The mevinolin effect is mediated through an increased rate of removal of rabbit 125I-labeled LDL from plasma. To study the role of mevinolin on the regulation of the hepatic LDL receptor in rabbits, the binding of 125I-labeled LDL and 125I-labeled beta-VLDL (beta-migrating very-low-density lipoproteins) to liver membranes prepared from rabbits fed the wheat starch-casein diet with or without mevinolin was investigated. Liver membranes from wheat starch-casein-fed rabbits have no demonstrable EDTA-sensitive binding activity of 125I-labeled LDL and low (37 ng/mg protein) binding activity of 125I-labeled beta-VLDL. Treatment of the wheat starch-casein fed rabbits with mevinolin results in high levels of specific EDTA-sensitive binding of 125I-labeled LDL (28.7 ng/mg protein) and 125I-labeled beta-VLDL (120 ng/mg protein). To assess the functional role of the hepatic LDL receptor in response to mevinolin, the catabolism of 125I-labeled LDL by perfused rabbit livers was studied. Perfused livers from mevinolin-treated rabbits show a 3.3-fold increase in the rate of receptor-dependent catabolism of 125I-labeled LDL (4.6% X h-1) when compared with that of livers from rabbits not treated with mevinolin (1.4% X h-1). Thus, these studies demonstrate that mevinolin prevents the increase of plasma LDL cholesterol level in rabbits fed a wheat starch-casein diet by regulating the levels of hepatic LDL-binding sites and the rate of receptor-dependent catabolism of LDL by the liver.     

Loci of catabolism of beta-very low density lipoprotein in vivo delineated with a residualizing label, 125I-dilactitol tyramine

beta-Very low density lipoprotein (beta-VLDL) may be a major atherogenic lipoprotein, and knowledge of the sites of its catabolism should facilitate elucidation of mechanisms important in the regulation of its plasma concentrations. In this study, catabolic sites of beta-VLDL have been delineated in normolipidemic rabbits with a novel, radioiodinated, residualizing label, 125I-dilactitol tyramine (125I-DLT). Comparative studies of beta-VLDL and low density lipoprotein catabolism were performed with 125I-DLT conjugated to each lipoprotein and with lipoproteins iodine-labeled conventionally. Conjugation did not alter size distributions or charge characteristics of lipoprotein particles. The overall processing (binding and degradation) of lipoproteins by cultured rabbit skin fibroblasts was not influenced by 125I-DLT derivatization, suggesting that attachment of the label did not influence cell receptor-lipoprotein interactions. Furthermore, although degradation products of 125I-lipoproteins leaked out of the cells and into the medium, the degradation products of 125I-DLT lipoproteins were retained by the cells. The principal catabolic site of beta-VLDL in normolipidemic rabbits was found to be the liver with 54 +/- 4% of injected 125I retained in this organ 24 h after injection of 125I-DLT-beta-VLDL. When catabolism was normalized to tissue weight, the liver and adrenals were found to be approximately equally active in the metabolism of beta-VLDL. In agreement with results of other studies with residualizing labels, the principal organ of catabolism of 125I-DLT-LDL in vivo was the liver. The adrenals were the most highly catabolizing organ when results were normalized for tissue weight. The quantitative differences observed in the tissue distributions of injected 125I-DLT-beta-VLDL and 125I-DLT-low density lipoprotein suggested that a significant proportion of beta-VLDL is removed by tissues before conversion to low density lipoprotein.     

The ligand-binding function of hepatic lipase modulates the development of atherosclerosis in transgenic mice

To investigate the separate contributions of the lipolytic versus ligand-binding function of hepatic lipase (HL) to plasma lipoprotein metabolism and atherosclerosis, we compared mice expressing catalytically active wild-type HL (HL-WT) and inactive HL (HL-S145G) with no endogenous expression of mouse apoE or HL (E-KO x HL-KO, where KO is knockout). HL-WT and HL-S145G reduced plasma cholesterol (by 40 and 57%, respectively), non-high density lipoprotein cholesterol (by 48 and 61%, respectively), and apoB (by 36 and 44%, respectively) (p < 0.01), but only HL-WT decreased high density lipoprotein cholesterol (by 67%) and apoA-I (by 54%). Compared with E-KO x HL-KO mice, both active and inactive HL lowered the pro-atherogenic lipoproteins by enhancing the catabolism of autologous (125)I-apoB very low density/intermediate density lipoprotein (VLDL/IDL) (fractional catabolic rates of 2.87 +/- 0.04/day for E-KO x HL-KO, 3.77 +/- 0.03/day for E-KO x HL-WT, and 3.63 +/- 0.09/day for E-KO x HL-S145G mice) and (125)I-apoB-48 low density lipoprotein (LDL) (fractional catabolic rates of 5.67 +/- 0.34/day for E-KO x HL-KO, 18.88 +/- 1.72/day for E-KO x HL-WT, and 9.01 +/- 0.14/day for E-KO x HL-S145G mice). In contrast, the catabolism of apoE-free, (131)I-apoB-100 LDL was not increased by either HL-WT or HL-S145G. Infusion of the receptor-associated protein (RAP), which blocks LDL receptor-related protein function, decreased plasma clearance and hepatic uptake of (131)I-apoB-48 LDL induced by HL-S145G. Despite their similar effects on lowering pro-atherogenic apoB-containing lipoproteins, HL-WT enhanced atherosclerosis by up to 50%, whereas HL-S145G markedly reduced aortic atherosclerosis by up to 96% (p < 0.02) in both male and female E-KO x HL-KO mice. These data identify a major receptor pathway (LDL receptor-related protein) by which the ligand-binding function of HL alters remnant lipoprotein uptake in vivo and delineate the separate contributions of the lipolytic versus ligand-binding function of HL to plasma lipoprotein size and metabolism, identifying an anti-atherogenic role of the ligand-binding function of HL in vivo.     

Comparison of plasma clearance of low density lipoprotein with beta-very low density lipoprotein or acetoacetylated low density lipoprotein in cholesterol-fed rabbits

The plasma clearance and tissue distribution of radioiodinated low-density lipoprotein (LDL), beta-very low density lipoprotein (beta-VLDL), and acetoacetylated LDL were studied in cholesterol-fed rabbits. Radioiodinated LDL ([125I]LDL) was cleared more slowly than either [125I]beta-VLDL or acetoacetylated-[125I]LDL and its fractional catabolic rate was one-half that of [125I]beta-VLDL and one-ninth that of acetoacetylated-[125I]LDL. Forty-eight hours after the injection of the labeled lipoproteins, the hepatic uptake was the greatest among the organs evaluated with the uptake of [125I]LDL being one-third that of either [125I]beta-VLDL or acetoacetylated-[125I]LDL. The reduction in the hepatic uptake of LDL due to a down-regulation of the receptors would account for this retarded plasma clearance.     

Hepatic low density lipoprotein receptor binding and lipid profile in Mastomys natalensis during Plasmodium berghei infection

Plasmodium berghei infection to Mastomys natalensis showed hyper beta-lipoproteinemia. The increase in serum cholesterol is associated with decreased uptake of low density lipoprotein (LDL) by the liver through receptor mediated endocytosis. The membranes prepared from infected M. natalensis exhibit up to 50% decline in high affinity binding sites for human 125I-LDL. Significant increases in serum lipids, cholesterol, triglyceride and lipid peroxide (LPO) contents of liver membrane were observed. Effects of lipid constituents and LPO content of liver membrane in relation to LDL catabolism and other possible mechanisms have been explained.     

Effect of insulin deficiency on low density lipoprotein metabolism in rabbits

These studies have been carried out in rabbits with alloxan-induced diabetes in order to see if insulin deficiency affects low density lipoprotein (LDL) catabolism. The results showed that plasma LDL-cholesterol was lower in diabetic rabbits, associated with a fall in the cholesterol to protein ratio of LDL particles. In addition, 125I-LDL disappeared more slowly from plasma of diabetic rabbits, leading to a significant reduction in fractional catabolic rate and a decrease in residence time of 125I-LDL. These data demonstrated that LDL composition and catabolism are greatly altered as a consequence of insulin deficiency.     

Glycosylation of LDL decreases its ability to interact with high-affinity receptors of human fibroblasts in vitro and decreases its clearance from rabbit plasma in vivo

Incubation of human LDL in vitro at 37 degrees C for 48 h with [14C]glucose at concentrations from 5 to 200 mM resulted in a glycosylated LDL, containing 0.4-20 mol of glucose incorporated per apolipoprotein B of 250 000 daltons. The extent of glucose incorporated was proportional to the time of incubation and concentration of glucose. Glycosylation of LDL abolished its uptake and degradation by the high-affinity process for LDL in normal human skin fibroblasts. 125I-labeled glycosylated LDL was bound, internalized and degraded by the fibroblasts via a nonspecific low-affinity process. The 125I-labeled glycosylated LDL and 125I-labeled LDL were taken up and degraded at similar rates in a non-saturable, low-affinity process by peritoneal macrophages isolated from mice. When 125I-labeled glycosylated LDL or 125I-labeled LDL were injected into rabbits, the glycosylated LDL had a delayed plasma clearance in comparison to the LDL. The mean fractional catabolic rates were 0.67 day-1 and 1.70 day-1 for 125I-labeled glycosylated LDL and 125I-labeled LDL, respectively. The uptake and degradation of 125I-labeled LDL by human skin fibroblasts was decreased as the concentration of free carbohydrate, glucose, sucrose or sorbitol, in the medium was increased from 10 mM to 1 M. It is speculated that pathologic levels of plasma glucose in vivo could result in a decrease in LDL uptake as a result of glycosylation of LDL. A decrease in uptake of native or modified LDL in vivo could contribute to hypercholesterolemia and its pathophysiology.     

Low density lipoprotein binding, internalization, and degradation in human adipose cells.

Human adipose tissue derives its cholesterol primarily from circulating lipoproteins. To study fat cell-lipoprotein interactions, low density lipoprotein (LDL) uptake and metabolism were examined using isolated human adipocytes. The 125I-labelled LDL (d = 1.025-1.045) was bound and incorporated by human fat cells in a dose-dependent manner with an apparent Km of 6.9 + 0.9 microgram LDL protein/mL and a Vmax of 15-80 microgram LDL protein/mg lipid per 2 h. In time-course studies, LDL uptake was characterized by rapid initial binding followed by a linear accumulation for at least 4 h. The 125I-labelled LDL degradation products (trichloroacetic acid soluble iodopeptides) accumulated in the incubation medium in a progressive manner with time. Azide and F- inhibited LDL internalization and degradation, suggesting that these processes are energy dependent. Binding and cellular internalization of 125I-labelled LDL lacked lipoprotein class specificity in that excess (25-fold) unlabelled very low density lipoprotein (VLDL) (d less than 1.006) and high density lipoprotein (HDL) (d = 1.075-1.21) inhibited binding and internalization of 125I-labelled LDL. On an equivalent protein basis HDL was the most potent. The 125I-labelled LDL binding to an adipocyte plasma membrane preparation was a saturable process and almost completely abolished by a three- to four-fold greater concentration of HDL. The binding, internalization, and degradation of LDL by human adipocytes resembled that reported by other mesenchymal cells and could account for a significant proportion of in vivo LDL catabolism. It is further suggested that adipose tissue is an important site of LDL and HDL interactions.     

Dietary restriction amplifies the metabolic disturbances of very-low-density lipoproteins in cholesterol-fed rabbits

The effect of dietary restriction (half of the control ration) on VLDL turnover was investigated in cholesterol-fed rabbits. Rabbits on standard, cholesterol and restricted cholesterol diets were injected with homologous 125I-labelled VLDL. Accompanying the amplification of hypercholesterolemia, additional disturbances of VLDL turnover were observed when cholesterol feeding was associated with dietary restriction. Cholesterol-fed rabbits with normal caloric ration exhibited delayed clearance of 125I-labelled apolipoprotein B component of VLDL compared to control rabbits. This was markedly accentuated in underfed rabbits, indicating further down-regulation of apolipoprotein B,E receptors in these animals. Furthermore, a reduced proportion of radiolabelled apolipoprotein B was converted from VLDL to intermediate-density lipoprotein (IDL) and LDL in both groups receiving cholesterol-rich diets. Thus, the combination of further impairment in plasma clearance of VLDL and the poor conversion into IDL and LDL could account for the massive increase of beta-VLDL in underfed animals on cholesterol-rich diets.     

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.     

Human monocyte colony-stimulating factor enhances the clearance of lipoproteins containing apolipoprotein B-100 via both low density lipoprotein receptor-dependent and -independent pathways in rabbits

To investigate the effects of recombinant human monocyte colony-stimulating factor (M-CSF) on plasma cholesterol metabolism, we injected M-CSF intravenously into New Zealand White rabbits (n = 13) at a dose of 100 micrograms/day for 7 days. After the treatment, the plasma cholesterol levels fell by 33.2% from 61.4 +/- 25.9 to 41.0 +/- 10.2 mg/dl (mean +/- S.D.). We also injected a large dose of M-CSF (500 micrograms/day) for 6 days into Watanabe Heritable Hyperlipidemic rabbits, which are deficient in low density lipoprotein (LDL) receptors. Again, there was a significant reduction in plasma cholesterol levels by 36.2% from 730.5 +/- 176.4 to 466.0 +/- 104.9 mg/dl (n = 4). In the kinetic studies in New Zealand White rabbits with very low density lipoprotein, LDL, and methylated LDL, the removal rates of those lipoproteins were increased 1.9-, 1.7-, and 2.0-fold, respectively, after the treatment. Immunoblot analysis of LDL receptors in the treated rabbits showed no significant changes in LDL receptor proteins in livers but a great increase in spleens and bone marrows compared with the controls. Messenger RNA was also estimated by Northern blotting in both groups, and the results were compatible with those from the immunoblot. The data suggest that M-CSF stimulates the clearance of lipoproteins containing apolipoprotein B-100 via both LDL receptor-dependent and -independent pathways in target cells of M-CSF and reduces plasma cholesterol.     

Age-related decreases in tissue sterol acquisition are mediated by changes in cholesterol synthesis and not low density lipoprotein uptake in the rat

The present investigation compared plasma cholesterol levels and lipoprotein profiles, and absolute rates of sterol synthesis and low density lipoprotein (LDL) uptake in various organs of immature (4 weeks old) and mature (15 weeks) rats. The plasma cholesterol level and its distribution among the major lipoprotein density fractions were similar in both groups. Using [3H]water as a substrate for measuring sterol synthesis in vivo, the content of newly synthesized cholesterol (3H-labeled digitonin-precipitable sterols; [3H]DPS) was several fold higher in all tissues of the young, compared to the old, rats when normalized per g of tissue. In contrast, whole-body [3H]DPS content was identical at 29.5 and 29.3 mumol/hr in young and old rats, respectively, despite a 4.4-fold difference in body weight (102 vs. 453 g). The importance of different organs to total-body sterol synthesis remained similar with increasing age although the skin (11 vs. 24% of total) rather than the small bowel (15 vs. 8%) became the second most important organ after the liver (49 vs. 45%) in the older animals. When LDL uptake was determined in these same organs, using constant infusion technique, the rates of clearance were higher only in the adrenal glands, adipose tissue, and skin of the young animals; whereas these rates were essentially the same in the liver and gastrointestinal tract, the two organs that are quantitatively most important for LDL catabolism. Even when these clearance rates were normalized to the whole organ or to 100 g of body weight, the differences in LDL uptake in the two age groups were minor compared to the major decrease in rates of cholesterol synthesis that were observed with aging. Finally, calculation of absolute rates of tissue cholesterol acquisition from both sources indicated that, in most organs, the majority of tissue cholesterol was derived from local synthesis rather than from LDL uptake in both age groups and that, with increasing age, total cholesterol acquisition decreased several-fold primarily as a consequence of the diminished rate of sterol synthesis. These studies demonstrate that with growth and aging in the rat there is a dramatic decrease in the rate of tissue cholesterol synthesis while the uptake of LDL-cholesterol remains essentially unchanged.