Evidence that chylomicron remnants and beta-VLDL are transported by the same receptor pathway in J774 murine macrophage-derived cells

To characterize lipoprotein uptake by macrophages, we studied J774 murine macrophage-derived cells. Uptake of 125I-labeled beta-VLDL and 125I-labeled chylomicron remnants was saturable, specific, and of high affinity. Maximal specific uptake and the concentration at which half-maximal uptake occurred were similar for both beta-VLDL and chylomicron remnants. Specific uptake of 125I-labeled chylomicrons was only 1/5 that of the other two lipoproteins. Cholesterol loading decreased 125I-labeled chylomicron remnant and 125I-labeled beta-VLDL uptake by 25%. Chylomicron remnants and beta-VLDL were equipotent in cross-competition studies; acetyl-LDL did not compete, and human LDL was a poor competitor. Although the amounts of cell-associated lipoproteins were similar, beta-VLDL and chylomicron remnants had different effects on cellular lipid metabolism. beta-VLDL produced a threefold stimulation while chylomicron remnants caused a decrease in [3H]oleate incorporation into cholesteryl ester. beta-VLDL had no effect while chylomicron remnants caused a threefold increase in [3H]oleate incorporation into triacylglycerol. beta-VLDL produced a 44% suppression and chylomicron remnants produced a 78% increase in HMG-CoA reductase activity. In summary, J774 macrophages express a receptor site that recognizes both beta-VLDL and chylomicron remnants; however, these lipoproteins exhibit strikingly different effects on intracellular lipid metabolism.     

Differences in the processing of chylomicron remnants and beta-VLDL by macrophages

To gain a detailed understanding of those factors that govern the processing of dietary-derived lipoprotein remnants by macrophages we examined the uptake and degradation of rat triacylglycerol-rich chylomicron remnants and rat cholesterol-rich beta-very low density lipoprotein (beta-VLDL) by J774 cells and primary cultures of mouse peritoneal macrophages. The level of cell associated 125I-labeled beta-VLDL and 125I-labeled chylomicron remnants reached a similar equilibrium level within 2 h of incubation at 37 degrees C. However, the degradation of 125I-labeled beta-VLDL was two to three times greater than the degradation of 125I-labeled chylomicron remnants at each time point examined, with rates of degradation of 161.0 +/- 36.0 and 60.1 +/- 6.6 ng degraded/h per mg cell protein, respectively. At similar extracellular concentrations of protein or cholesterol, the relative rate of cholesteryl ester hydrolysis from [3H]cholesteryl oleate/cholesteryl [14C]oleate-labeled chylomicron remnants was one-third to one-half that of similarly labeled beta-VLDL. The reduction in the relative rate of chylomicron remnant degradation by macrophages occurred in the absence of chylomicron remnant-induced alterations in low density lipoprotein (LDL) receptor recycling or in retroendocytosis of either 125I-labeled lipoprotein. The rate of internalization of 125I-labeled beta-VLDL by J774 cells was greater than that of 125I-labeled chylomicron remnants, with initial rates of internalization of 0.21 ng/min per mg cell protein for 125I-labeled chylomicron remnants and 0.39 ng/min per mg cell protein for 125I-labeled beta-VLDL. The degradation of 125I-labeled chylomicron remnants and 125I-labeled beta-VLDL was dependent on lysosomal enzyme activity: preincubation of macrophages with the lysosomotropic agent monensin reduced the degradation of both lipoproteins by greater than 90%. However, the pH-dependent rate of degradation of 125I-labeled chylomicron remnants by lysosomal enzymes isolated from J774 cells was 50% that of 125I-labeled beta-VLDL. The difference in degradation rates was dependent on the ratio of lipoprotein to lysosomal protein used and was greatest at ratios greater than 50. The degradation of 125I-labeled beta-VLDL by isolated lysosomes was reduced 30-40% by preincubation of beta-VLDL with 25-50 micrograms oleic acid/ml, suggesting that released free fatty acids could cause the slower degradation of chylomicron remnants. Thus, differences in the rate of uptake and degradation of remnant lipoproteins of different compositions by macrophages are determined by at least two factors: 1) differences in the rates of lipoprotein internalization and 2) differences in the rate of lysosomal degradation.     

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.     

Regulation of the uptake and degradation of beta-very low density lipoprotein in human monocyte macrophages

In normal human monocyte macrophages 125I-labeled beta-migrating very low density lipoproteins (125I-beta-VLDL), isolated from the plasma of cholesterol-fed rabbits, and 125I-human low density lipoprotein (LDL) were degraded at similar rates at protein concentrations up to 50 micrograms/ml. The high affinity degradation of 125I-labeled human LDL saturated at approximately 50 micrograms/ml; however, 125I-labeled rabbit beta-VLDL high affinity degradation saturated at 100-120 micrograms/ml. The activity of the beta-VLDL receptor was 3-fold higher than LDL receptor activity on freshly isolated normal monocyte macrophages, but with time-in-culture both receptor activities decreased and were similar after several days. The degradations of both beta-VLDL and LDL were Ca2+ sensitive, were markedly down regulated by sterols, and were up regulated by preincubation of the cells in a lipoprotein-free medium. The beta-VLDL receptor is genetically distinct from the LDL receptor as indicated by its presence on monocyte macrophages from a familial hypercholesterolemic homozygote. Human thoracic duct lymph chylomicrons as well as lipoproteins of Sf 20-5000 from fat-fed normal subjects inhibited the degradation of 125I-labeled rabbit beta-VLDL as effectively as nonradioactive rabbit beta-VLDL. We conclude: 1) the beta-VLDL receptor is genetically distinct from the LDL receptor, and 2) intestinally derived human lipoproteins are recognized by the beta-VLDL receptor on macrophages.     

Regulation of the hepatic removal of chylomicron remnants and beta-very low density lipoproteins in the rat.

The contribution of the low density lipoprotein (LDL) receptor to the removal of chylomicron remnants was determined in vitro and in vivo by using interventions that up- or down-regulate the LDL receptor but not the LDL receptor-related protein (LRP). In vitro, chylomicron remnants and beta-very low density lipoprotein (VLDL) bind to the LDL receptor on endosomal membranes; their binding can be competed by LDL and beta-VLDL and the binding capacity is greatly augmented in membranes from estradiol-treated rats. Likewise, estradiol treatment almost doubled the removal of chylomicron remnants during a single pass through perfused rat livers. However, in vivo the removal of chylomicron remnants and beta-VLDL was very rapid even in untreated rats so that the effect of the stimulation by estradiol was barely detectable when trace amounts of lipoproteins were injected. Yet, when saturating doses of either lipoprotein were injected, the effect of estradiol treatment on the removal of chylomicron remnants and beta-VLDL was readily disclosed. In rats fed a diet containing lard, cholesterol, and bile acids, removal of chylomicron remnants or beta-VLDL was significantly retarded. Likewise, perfused livers from diet-fed rats removed only a mean of 16% of chylomicron remnants during a single passage as compared to 29% in livers from control animals. Also, when large doses of beta-VLDL had been infused into rats for 4 h, in subsequent perfusions of the livers the removal of chylomicron remnants was decreased to 11%. From these results it is concluded that the LDL receptor mediates the hepatic removal of a major fraction of chylomicron remnants and beta-VLDL.     

Transport of beta-very low density lipoproteins and chylomicron remnants by macrophages is mediated by the low density lipoprotein receptor pathway

The receptor-mediated uptake of rat hypercholesterolemic very low density lipoproteins (beta VLDL) and rat chylomicron remnants was studied in monolayer cultures of the J774 and P388D1 macrophage cell lines and in primary cultures of mouse peritoneal macrophages. Uptake of 125I-beta VLDL and 125I-chylomicron remnants was reduced 80-90% in the presence of high concentrations of unlabeled human low density lipoproteins (LDL). Human acetyl-LDL did not significantly compete at any concentration tested. Uptake of 125I-beta VLDL and 125I-chylomicron remnants was also competitively inhibited by specific polyclonal antibodies directed against the estrogen-induced LDL receptor of rat liver. Incubation in the presence of anti-LDL receptor IgG, but not nonimmune IgG, reduced specific uptake greater than 80%. Anti-LDL receptor IgG, 125I-beta VLDL, and 125I-chylomicron remnants bound to two protein components of apparent molecular weights 125,000 and 111,000 on nitrocellulose blots of detergent-solubilized macrophage membranes. Between 70-90% of 125I-lipoprotein binding was confined to the 125,000-Da peptide. Binding of 125I-beta VLDL and 125I-chylomicron remnants to these proteins was competitively inhibited by anti-LDL receptor antibodies. Comparison of anti-LDL receptor IgG immunoblot profiles of detergent-solubilized membranes from mouse macrophages, fibroblasts, and liver, and normal and estrogen-induced rat liver demonstrated that the immunoreactive LDL receptor of mouse cells is of a lower molecular weight than that of rat liver. Incubation of J774 cells with 1.0 micrograms of 25-hydroxycholesterol/ml plus 20 micrograms of cholesterol/ml for 48 h decreased 125I-beta VLDL uptake and immuno- and ligand blotting to the 125,000- and 111,000-Da peptides by only 25%. Taken together, these data demonstrate that uptake of beta VLDL and chylomicron remnants by macrophages is mediated by an LDL receptor that is immunologically related to the LDL receptor of rat liver.     

Uptake of canine beta-very low density lipoproteins by mouse peritoneal macrophages is mediated by a low density lipoprotein receptor

The receptor on mouse peritoneal macrophages that mediates the uptake of canine beta-very low density lipoproteins (beta-VLDL) has been identified in this study as an unusual apolipoprotein (apo-) B,E(LDL) receptor. Ligand blots of Triton X-100 extracts of mouse peritoneal macrophages using 125I-beta-VLDL identified a single protein. This protein cross-reacted with antibodies against bovine apo-B,E(LDL) receptors, but its apparent Mr was approximately 5,000 less than that of the human apo-B,E(LDL) receptor. Binding studies at 4 degrees C demonstrated specific and saturable binding of low density lipoproteins (LDL), beta-VLDL, and cholesterol-induced high density lipoproteins in plasma that contain apo-E as their only protein constituent (apo-E HDLc) to mouse macrophages. Apolipoprotein E-containing lipoproteins (beta-VLDL and apo-E HDLc) bound to mouse macrophages and human fibroblasts with the same high affinity. However, LDL bound to mouse macrophages with an 18-fold lower affinity than to human fibroblasts. Mouse fibroblasts also bound LDL with a similar low affinity. Compared with the apo-B,E(LDL) receptors on human fibroblasts, the apo-B,E(LDL) receptors on mouse macrophages were resistant to down-regulation by incubation of the cells with LDL or beta-VLDL. There are three lines of evidence that an unusual apo-B,E(LDL) receptor on mouse peritoneal macrophages mediates the binding and uptake of beta-VLDL: LDL with residual apo-E removed displaced completely the 125I-beta-VLDL binding to mouse macrophages, preincubation of the mouse macrophages with apo-B,E(LDL) receptor antibody inhibited both the binding of beta-VLDL and LDL to the cells and the formation of beta-VLDL- and LDL-induced cholesteryl esters, and binding of 125I-beta-VLDL to the cells after down-regulation correlated directly with the amount of mouse macrophage apo-B,E(LDL) receptor as determined on immunoblots. This unusual receptor binds LDL poorly, but binds apo-E-containing lipoproteins with normal very high affinity and is resistant to down-regulation by extracellular cholesterol.     

Effects of chylomicron remnants and beta-VLDL on the class and composition of newly secreted lipoproteins by HepG2 cells

The regulation of lipoprotein secretion in the cell line HepG2 was studied. HepG2 cells were preincubated with chylomicron remnants (triglyceride- and cholesterol-rich) or with beta very low density lipoproteins (beta-VLDL) (cholesterol-rich). The medium was removed and the cells were incubated for and additional 24 hr in a lipoprotein-free medium that contained either [2-3H]glycerol or DL-[2-3H]mevalonate. Cells and media were harvested, and lipoproteins were separated and fractionated. The mass and radioactivity of the lipids in cells and in the lipoproteins were measured. The activities of cellular acyl-CoA:cholesterol acyltransferase (ACAT) and 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase were also determined. Preincubation with chylomicron remnants induced an increase in cellular triglyceride and stimulated both HMG-CoA reductase and ACAT. Preincubation with beta-VLDL induced an increase in cellular free and esterified cholesterol, inhibited HMG-CoA reductase and stimulated ACAT. Although the absolute amount of VLDL is small, chylomicron remnants induced large relative increases in the amount of triglyceride and phospholipid secreted in VLDL and decreases in the amount of triglyceride secreted in low density (LDL) and high density (HDL) lipoproteins as well as a decrease in the amount of phospholipid secreted in HDL. In contrast, preincubation with beta-VLDL did not affect triglyceride secretion, but markedly stimulated the amount of phospholipid secreted in HDL. Comparison of the mass of glycerolipid actually secreted with that calculated from the cellular specific activity suggested that glycerolipids are secreted from single, rapidly equilibrating pools. Cholesterol and cholesteryl ester secretion were affected differently. Preincubation with chylomicron remnants increased the amount of free cholesterol secreted in both VLDL and LDL, but did not alter cholesteryl ester secretion. Preincubation with beta-VLDL increased free cholesterol secretion in all lipoprotein fractions and increased cholesteryl ester secretion in VLDL and LDL, but not HDL. Comparison of isotope and mass data suggested that the cholesteryl ester secreted came primarily from a preformed, rather than an newly synthesized, pool. In summary, these data provide insight to the mechanism whereby a liver cell regulates the deposition of exogenous lipid.     

Characterization of lipoprotein receptors on rat Fu5AH hepatoma cells

The rat hepatoma cell line Fu5AH has the unusual property of accumulating massive amounts of cholesteryl ester upon incubation with hypercholesterolemic serum, and especially when incubated with beta-very low density lipoproteins (beta-VLDL) from cholesterol-fed dogs. The present study was designed to identify and characterize the lipoprotein receptors that mediate the cholesteryl ester accumulation. The beta-VLDL and cholesterol-induced apolipoprotein (apo) E-containing high density lipoproteins (apoE HDLc) bound to Fu5AH cells with very high affinity (Kd approximately equal to 10(-10) M), whereas low density lipoproteins (LDL) bound with unusually low affinity (Kd approximately equal to 10(-8) M). Receptor binding activity of 125I-labeled beta-VLDL, 125I-labeled apoE HDLc, and 125I-labeled LDL was abolished by incubation in the presence of an excess of unlabeled LDL or of a polyclonal antibody to the bovine adrenal apoB,E(LDL) receptor. The receptors were completely down-regulated by preincubating Fu5AH cells with beta-VLDL, but much higher levels of beta-VLDL were required than for down-regulation of fibroblast apoB,E(LDL) receptors. Receptor binding was abolished by reductive methylation of the lysyl residues of the apolipoprotein of the beta-VLDL and by an apoE monoclonal antibody (1D7) that blocks receptor binding. The Fu5AH receptor was further characterized by using the bovine adrenal apoB,E(LDL) receptor antibody. A single protein (Mr approximately equal to 130,000) was identified in Triton extracts of whole cells, and two proteins (Mr approximately equal to 130,000 and 115,000) were found in Fu5AH cell membranes disrupted by homogenization. The Mr approximately equal to 115,000 protein was released from the membranes and did not react with an antibody to the carboxyl-terminal (cytoplasmic) domain of the apoB,E(LDL) receptors. These studies indicate that Fu5AH cells express apoB,E(LDL) receptors that have unusually low affinity for apoB-continuing lipoproteins, require large amounts of cholesterol to induce down-regulation, and are susceptible to specific proteolysis in cell homogenates. These apoB,E(LDL) receptors are responsible for the receptor-mediated uptake of beta-VLDL and chylomicron remnants by Fu5AH cells.     

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.     

Clearance of chylomicron remnants by the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor.

The involvement of the low density lipoprotein receptor-related protein (LRP) in chylomicron remnant (CR) catabolism was investigated. Ligand blot analyses demonstrated that beta-very low density lipoproteins (beta-VLDL) incubated with apolipoprotein E (beta-VLDL+E) bound to the LRP and low density lipoprotein receptors, whereas active (receptor-binding) alpha 2-macroglobulin (alpha 2M) bound only to LRP partially purified from rat liver membranes. Iodinated beta-VLDL+E and active alpha 2M showed high affinity binding to the LRP/alpha 2M receptor of low density lipoprotein receptor-negative fibroblasts. The binding and degradation of radiolabeled alpha 2M by these cells were partially inhibited by beta-VLDL+E. Furthermore, alpha 2M interfered with the internalization of beta-VLDL+E and subsequent induction in the cholesterol esterification by these cells. These studies suggested that remnant lipoproteins and active alpha 2M compete for binding to the LRP/alpha 2M receptor. Next, we examined whether the LRP/alpha 2M receptor plays a role, in the presence of low density lipoprotein receptors, in the in vivo catabolism of CR in mice. In vivo studies demonstrated that the unlabeled active, but not the native, alpha 2M partially inhibited the plasma clearance and hepatic uptake of radiolabeled CR or apoE-enriched radiolabled CR. Likewise, apoE-enriched CR retarded the plasma clearance and hepatic uptake of radiolabeled active alpha 2M. These studies provide physiological evidence that the LRP/alpha 2M receptor may function as a CR receptor that removes CR from the plasma.     

Receptor-dependent uptake of human chylomicron remnants by cultured skin fibroblasts

Human chylomicrons were isolated from plasma from a subject with familial hypertriglyceridemia and converted to chylomicron remnants by incubation with postheparin plasma. The interaction of these apolipoprotein E-containing, cholesterol-rich human chylomicron remnants with cultured skin fibroblasts was studied. Chylomicron remnants were internalized by skin fibroblasts as a unit, mainly via the low density lipoprotein (LDL)-receptor pathway, resulting in increased cell cholesterol content. After entering the fibroblast, chylomicron remnants stimulated cholesterol esterification, suppressed 3-hydroxy-3-methylglutaryl coenzyme A reductase activity, and down-regulated LDL receptor activity similar to the action of LDL. As a function of increasing lipolysis, remnant particles were progressively more effectively taken up by skin fibroblasts, despite a decrease in the apolipoprotein E content per lipoprotein particle. Remnant particles produced after hydrolysis of 70 to 80% of chylomicron triglyceride increased cell cholesterol content to an amount nearly identical to that observed with LDL when the two lipoproteins were incubated at an equal cholesterol concentration. However, when incubated on the basis of equal particle number, chylomicron remnants were 2 to 3 times more effective than LDL in delivering cholesterol to the cells. These results suggest that chylomicron remnants play a role in the regulation of postabsorptive cholesterol homeostasis in nonhepatic cells, and possibly in the pathogenesis of atherosclerosis.     

Failure of the intracellular itinerary of beta very low density lipoproteins to augment cholesterol esterification in macrophages from Watanabe heritable hyperlipidemic rabbits

beta very low density lipoproteins (beta-VLDL) interact with mouse peritoneal macrophages via specific receptors leading to pronounced stimulation of cholesterol esterification. The present study has defined an alternative pathway for the processing of beta-VLDL in alveolar macrophages from Watanabe heritable hyperlipidemic (WHHL) rabbits. Macrophages from either New Zealand (NZ) or WHHL rabbits degraded 125I-beta-VLDL to an equivalent extent. Degradation was competed to a similar extent in both cell types by either excess unlabeled beta-VLDL or low density lipoprotein, indicative of a specific receptor involvement. Accumulation of intracellular degradation products of beta-VLDL labeled with the residualizing label, dilactitol-125I-tyramine, was similar in both cell types demonstrating that degradation was not due to secreted proteolytic enzymes. beta-VLDL promoted the incorporation of [3H]oleate into cholesteryl-[3H]oleate and increased the cellular mass of cholesterol in NZ macrophages. In contrast, beta-VLDL did not augment cholesteryl-[3H]oleate deposition in WHHL macrophages. This lack of cholesterol esterification occurred despite equivalent acyl-CoA:cholesterol acyltransferase activity in microsomal fractions of both cell types, and similar augmentations in cholesteryl-[3H]oleate during incubation with phospholipase C-treated LDL. Incubation of WHHL macrophages with beta-VLDL increased cellular cholesterol mass, although the response was attenuated compared to NZ cells. To determine whether these disparities in cholesterol esterification were related to the catabolic fate of beta-VLDL-derived cholesterol esters, [3H]cholesteryl oleate was exchanged into the core of beta-VLDL and incubated with macrophages in medium containing [14C]oleate. NZ macrophages accumulated both [3H]cholesterol and [3H]cholesteryl-[14C]oleate after 5 h, indicating hydrolysis and re-esterification of cholesterol esters. In contrast, WHHL macrophages only accumulated [3H]cholesterol esters, suggesting uptake of cholesterol esters without subsequent hydrolysis. These data demonstrate that WHHL macrophages possess a pathway for the intracellular processing of beta-VLDL that permits internalization of the particle without stimulation of cholesterol esterification.     

Opposing effects of apolipoproteins E and C on lipoprotein binding to low density lipoprotein receptor-related protein

The low density lipoprotein receptor-related protein (LRP) from rat liver membranes binds apoprotein E (apoE)-enriched rabbit beta-migrating very low density lipoproteins (beta-VLDL) in a ligand blotting assay on nitrocellulose membranes. Binding was markedly activated when the beta-VLDL was preincubated with recombinant human apoE-3, native human apoE-3 or E-4, or native rabbit apoE. Human apoE-2, which binds poorly (1-2% of apo E-3 binding) to low density lipoprotein receptors, was approximately 40% as effective as apoE-3 or apoE-4 in binding to LRP. Stimulation of apoE-dependent binding to LRP was blocked by the inclusion of a mixture of human apoC proteins, but not apoA-I or A-II, in the preincubation reaction. High concentrations of apoE did not overcome the apoC inhibition. The effects of apoE and apoC on the ligand blotting assay were paralleled by similar effects in the ability of beta-VLDL to stimulate cholesteryl ester synthesis in mutant human fibroblasts that lack low density lipoprotein receptors. These properties of LRP are consistent with the known effects of apoE and apoC on uptake of chylomicron and very low density lipoprotein remnants in the liver and raise the possibility that LRP functions as a receptor for apoE-enriched forms of these lipoproteins in intact animals.     

The beta-VLDL receptor pathway of murine P388D1 macrophages

Very low density lipoproteins Sf 100-400 (VLDL1) from hypertriglyceridemic (HTG) subjects and chylomicrons cause receptor-mediated lipid engorgement in unstimulated macrophages in vitro via the beta-VLDL receptor pathway. We now report that the murine macrophage P388D1 cell line possesses the characteristics of the beta-VLDL receptor pathway observed previously in freshly isolated resident murine peritoneal macrophages or human monocyte-macrophages. HTG-VLDL1 isolated from the plasma of subjects with hypertriglyceridemia types 3, 4, and 5 interact with P388D1 macrophages in a high-affinity, curvilinear manner. beta-VLDL, HTG-VLDL1, chylomicrons, and thrombin-treated HTG-VLDL1 (which do not bind to the LDL receptor) compete efficiently and similarly for the uptake and degradation of HTG-VLDL1. LDL and acetyl LDL do not compete, indicating that uptake of HTG-VLDL1 is via neither the LDL receptor nor the acetyl LDL receptor. Binding of thrombin-treated HTG-VLDL1 to the beta-VLDL receptor indicates that the thrombin-accessible apoE, which is absolutely required for interaction of HTG-VLDL Sf greater than 60 with the LDL receptor, is not required for binding to the beta-VLDL receptor. The uptake and degradation of 125I-labeled HTG-VLDL1 is suppressed up to 80-90% by preincubation of the cells with sterols, acetyl LDL, or beta-VLDL, indicating that this process is not via the irrepressible chylomicron remnant (apoE) receptor. Chylomicrons, HTG-VLDL1, and thrombin-treated HTG-VLDL1-but not normal VLDL1, beta-VLDL, LDL, or acetyl LDL-produce massive triglyceride accumulation (10-20-fold mass increases in 4 hr) in P388D1 macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distinct properties of the recognition sites for beta-very low density lipoprotein (remnant receptor) and alpha 2-macroglobulin (low density lipoprotein receptor-related protein) on rat parenchymal cells.

The properties of the recognition sites for alpha 2-macroglobulin (alpha 2-macroglobulin receptor; low density lipoprotein receptor-related protein) and beta-migrating very low density lipoprotein (beta-VLDL) (remnant receptor) on rat parenchymal cells were directly compared to analyze whether both substrates are recognized and internalized by the same receptor system. In cholesterol-fed rats, the large circulating pool of beta-VLDL is unable to diminish the liver uptake of 125I-labeled alpha 2-macroglobulin, while liver uptake of 125I-labeled beta-VLDL in these rats is reduced by 87.3% at 10 min after injection. In vitro competition studies with isolated parenchymal liver cells demonstrate that the binding of 125I-labeled alpha 2-macroglobulin to rat parenchymal cells is not effectively competed for by beta-VLDL, whether this lipoprotein is additionally enriched in apolipoprotein E or not. Binding of alpha 2-macroglobulin to parenchymal cells requires the presence of calcium, while binding of beta-VLDL does not. Incubation of parenchymal cells for 1 h with proteinase K reduced the subsequent binding of alpha 2-macroglobulin by 90.1%, while the binding of beta-VLDL was reduced by only 20.2%. In the presence of monensin, the association of alpha 2-macroglobulin to parenchymal cells at 2 h of incubation was reduced by 64.7%, while the association of beta-VLDL was not affected. Preincubation of parenchymal cells with monensin for 60 min at 37 degrees C reduced the subsequent binding of alpha 2-macroglobulin by 54.5%, while binding of beta-VLDL was only reduced by 14.6%. The results indicate that the recognition sites for alpha 2-macroglobulin and beta-VLDL on rat parenchymal cells do exert different properties and are therefore likely to reside on different molecules.     

Rapid initial removal of chylomicron remnants by the mouse liver does not require hepatically localized apolipoprotein E

Apolipoprotein E (apoE) is a ligand for the low density lipoprotein receptor (LDLR) and the low density lipoprotein receptor-related protein (LRP). The aim of the present study was to clarify the role of hepatically localized apoE in the rapid initial removal of chylomicron remnants by using the isolated perfused liver. Radiolabeled chylomicron remnants were perfused in a single nonrecirculating pass into the livers of C57BL/6J (wild-type) mice, apoE-knockout mice, and apoE/LDLR-knockout mice for a period of 20 min. Aliquots of the perfusate leaving the liver were collected at regular intervals and the rate of removal of radioactivity was determined. At a trace concentration of chylomicron remnants (0.05 microgram of protein per ml), wild-type mouse livers removed at a steady state of 50-55% of total chylomicron remnants perfused per pass; livers from apoE-knockout mice had the same capacity as wild-type mouse livers. When the concentration of remnants was increased to 12 microgram of protein per ml, a level at which it has been shown that LDL receptor and LRP are near saturation, the capacity of the wild-type mouse livers to remove chylomicron remnants was decreased to 10-25% per pass, confirming that the removal mechanisms were nearing saturation. However, instead of finding a greater reduction in the removal rates or impairment in chylomicron remnant removal, livers from apoE-knockout mice were just as efficient as those from wild-type mice in removing remnants. Livers of mice that lacked both apoE and the LDLR also had a similar rate of removal at relatively low remnant concentrations (0.05-0.5 microgram/ml), but had reduced capacity in removing remnants at a relatively high concentration (4-12 microgram/ml) of chylomicron remnants ( approximately 20% per pass). The rate of removal at these concentrations, however, was similar to that attributed to the LRP in previous studies. Chylomicron remnants, whose apolipoproteins were disrupted by trypsinization, were removed at a normal rate by wild-type mouse livers but there was almost no removal by apoE-knockout mouse livers. At higher concentrations, however, the removal of apolipoprotein-disrupted chylomicron remnants was decreased.Our present findings do not support the hypothesis that hepatically localized apoE is a critical factor in the rapid initial removal of chylomicron remnants by either of the major pathways but do suggest that hepatically localized apoE can be added to lipoproteins to accelerate their uptake, although this process may have a limited capacity to compensate for apoE deficiency on lipoproteins.     

Binding of chylomicron remnants and beta-very low density lipoproteins to hepatic and extrahepatic lipoprotein receptors. A process independent of apolipoprotein B48

The ability of apolipoprotein (apo-) B48 to interact with lipoprotein receptors was investigated using three different types of lipoproteins. First, canine chylomicron remnants, which contained apo-B48 as their primary apoprotein constituent, were generated by the hydrolysis of chylomicrons with milk lipoprotein lipase. These apo-B48-containing chylomicron remnants are deficient in apo-E and reacted very poorly with apo-E receptors on adult dog liver membranes and the low density lipoprotein (apo-B,E) receptors on human fibroblasts. Addition of normal human apo-E3 restored the receptor binding activity of these lipoproteins. Second, beta-very low density lipoproteins (beta-VLDL) from cholesterol-fed dogs were subfractionated into distinct classes containing apo-E along with either apo-B48 or apo-B100. Both classes bound to the apo-B,E and apo-E receptors. Their binding was almost completely mediated by apo-E, as evidenced by the ability of the anti-apo-E to inhibit the receptor interaction. Third, beta-VLDL from type III hyperlipoproteinemic patients were subfractionated by immunoaffinity chromatography into lipoproteins containing apo-E plus either apo-B48 or apo-B100. Both subfractions bound poorly to apo-B,E and apo-E receptors due to the presence of defective apo-E2. However, the residual binding of the apo-B48-containing and apo-B100-containing human beta-VLDL was inhibited by the anti-apo-E. After lipase hydrolysis, apo-B100 became a more prominant determinant responsible for mediating receptor binding to the apo-B,E receptor. By contrast, lipase hydrolysis did not increase the binding activity of the apo-B48-containing beta-VLDL. These results indicate that apo-B48 does not play a direct role in mediating the interaction of lipoproteins with receptors on fibroblasts or liver membranes.     

Intimal smooth muscle cells up-regulate beta-very low density lipoprotein-mediated cholesterol accumulation by enhancing beta-very low density lipoprotein uptake and decreasing cholesterol efflux

To clarify the mechanism of smooth muscle cell (SMC)-derived foam cell formation, we investigated beta-very low density lipoprotein (beta-VLDL) cholesterol metabolism in vascular medial SMCs (M-SMCs) from normal rabbits compared with intimal SMCs (I-SMCs) from normal rabbits fed a high-cholesterol diet and LDL receptor-deficient rabbits. For both types of I-SMCs, uptake of [3H]cholesteryl oleate labeled beta-VLDL increased 1.6 times and release of [3H]cholesterol decreased 40% compared with M-SMCs. M-SMCs took up part of the beta-VLDL through the LDL receptor but I-SMCs did not. mRNAs for the VLDL receptor and the LDL receptor relative with 11 ligand binding repeats were expressed at similar levels in all SMCs. M-SMCs expressed more LDL receptor-related protein than I-SMCs. Ligand blotting analysis revealed greater 125I-beta-VLDL binding to a 700-kDa protein in I-SMCs compared with M-SMCs. I-SMCs had higher activities of acid cholesterol esterase and acyl-CoA:cholesterol acyltransferase, and lower activity of neutral cholesterol esterase than M-SMCs in both the absence and the presence of beta-VLDL. These results indicate that I-SMCs accumulate more cholesteryl ester than M-SMCs by taking up more beta-VLDL and by effluxing less cholesterol.     

Glycosaminoglycan-lipoprotein interaction

Glycosaminoglycans (GAGs) bound to various proteoglycans (PGs) present in the cardiovascular system have been proposed to perform a wide range of functions. These include conferring viscoelastic properties; interacting with and modulating growth factors and enzymes; and as receptors and co-receptors in lipoprotein metabolism. Binding of apoB-100 lipoproteins, particularly low density lipoproteins (LDL), to GAGs of extracellular matrix PGs in arteries has been proposed to be an initiating event in development of atherosclerosis. This study was initiated with the aim of getting an overview of the binding patterns of different lipoprotein subclasses with individual GAG categories. We thus evaluated the interaction of lipoproteins with GAGs commonly found in the cardiovascular system using a gel mobility-shift assay developed for this purpose. The same procedure was used to measure lipoproteins binding to metabolically [(35)S]-labeled whole PGs prepared from three cell types, arterial smooth muscle cells, THP-1 macrophages and from HepG2 cells. The effect of GAG composition on PGs on lipoprotein binding was evaluated by enzymatic degradation of the carbohydrate chains. Heparan sulfate was found to bind beta very low density lipoproteins (beta-VLDL) and a chylomicron remnant model (beta-VLDL+apoE), but not LDL. Dermatan sulfate was found to bind LDL, but not beta-VLDL or the chylomicron remnant model. Chondroitin sulfate and heparin were found to bind all lipoproteins tested (LDL, beta-VLDL and beta-VLDL+apoE) although with different affinities. We can conclude that each lipoprotein subclass tested binds a specific assortment of the GAGs tested. The observations made contribute to the understanding of new and complex mechanisms by which carbohydrate and lipid metabolism may be linked.