Differential effects of wortmannin on the release of substance P and amino acids from the isolated spinal cord of the neonatal rat

Mice lacking hepatic lipase have been reported to express mild hyperlipidemia characterized by increased concentrations of large high density lipoproteins, but normal concentrations of lipoproteins containing apolipoprotein B. Whereas hepatic lipase has been implicated in the clearance and processing of chylomicron remnants in rats, no such defect was found in these mice. We have further characterized the abnormal lipoprotein phenotype in young hepatic lipase-deficient mice and have found more pronounced elevations of high density lipoproteins associated in particular with a 5-fold increase in plasma concentrations of apolipoprotein E. In addition, there was a reduction in the concentration of low density lipoproteins containing apolipoprotein B-100 and B-48 relative to precursor lipoproteins of lower density and a pronounced deficiency of apolipoprotein B-containing low density lipoproteins with density exceeding 1.029 g/mL. Conversion of radiolabeled rabbit intermediate density lipoproteins to low density lipoproteins was reduced by 6-fold as compared with wild-type mice. Although clearance of cholesteryl ester-labeled chylomicrons from the blood was unimpaired in the deficient mice, that of chylomicron remnants was reduced. Furthermore, endocytosis of chylomicron cholesteryl esters into liver cells occurred more rapidly than in wild-type mice. The unimpaired hepatic clearance of injected chylomicron particles in hepatic lipase-deficient mice may be the result of greater acquisition of apoE from high density lipoproteins during remnant formation. These studies thus demonstrate a critical role for mouse hepatic lipase in the formation of small, dense low density lipoproteins, as well as participation in the normal clearance and processing of chylomicron remnants.     

Acceleration of uptake of LDL but not chylomicrons or chylomicron remnants by cells that secrete apoE and hepatic lipase

ApoE is a ligand for the low density lipoprotein (LDL) receptor as well as for the LDL receptor-related protein (LRP). The enzyme hepatic lipase (HL) may also affect the uptake of lipoproteins by modifying their composition. We have tested the hypothesis that hepatic lipase and apoE can function as co-factors to alter the rate of lipoprotein uptake. Chinese hamster ovary (CHO) cells were transfected with cDNAs for rat hepatic lipase, human apoE or both HL and apoE. The secreted recombinant proteins were thoroughly characterized and had properties identical to the native proteins. Hepatic lipase and apoE were secreted at 0.17 and 1.25 micrograms/mg cell protein per hour, rates comparable to those in normal liver. 125I-labeled LDL, chylomicron remnants, or chylomicrons were added to media at concentrations near their Kd. In cells that secreted either apoE or hepatic lipase, or both apoE and hepatic lipase, LDL binding was significantly greater than with control cells (2.2-, 2-, 2-fold greater, respectively). Similar enhancement of LDL degradation was observed. In the presence of anti-LDL receptor antibodies, these values were reduced to control levels; thus, the enhanced uptake was mediated by the LDL receptor and not the LRP. The amount of LDL receptor protein, as judged by Western blotting, was similar in the various cell types. Incubation of control CHO cells with media from secreting transfected cells also increased the uptake of 125I-labeled LDL. Kinetic studies indicated that, in apoE-secreting cells, increased LDL binding is associated with a lower Kd and an unchanged Vmax as compared to the control cells; furthermore, when LDL were reisolated by column chromatography (but not by ultracentrifugation) from the incubations where apoE was being secreted, apoE was identified adherent to the LDL particles. Together, these results suggest that the effect is due to alteration of the lipoprotein and not the cell. In contrast, the uptake of 125I-labeled chylomicron remnants, and 125I-labeled chylomicrons was not greater in the transfected cells. Thus, in the amounts secreted by these cells, hepatic lipase and apoE do not convert chylomicrons to chylomicron remnants or alter the uptake of chylomicron remnants by either the LDL receptor or the LRP. The enhancement of LDL removal in cells that secrete hepatic lipase or apoE may help determine the amount of LDL removed by a particular tissue.     

Multiple processes are involved in the uptake of chylomicron remnants by mouse peritoneal macrophages

The processes responsible for the uptake of chylomicron remnants by macrophages were investigated using freshly isolated cells from low density lipoprotein (LDL) receptor, very low density lipoprotein (VLDL) receptor and apolipoprotein E knockout mice. In peritoneal macrophages from normal mice, the metabolism of chylomicron remnants was inhibited 40% by anti-LDL receptor antibody and 60% by a high concentration of receptor-associated protein (RAP). Together they reduced the amount processed by 70%. Digestion of cell proteoglycans decreased remnant degradation by 20% while the addition of acetyl-LDL had no effect. When LDL receptors were absent, the absolute rates of metabolism were less than that of normal cells and were not inhibited by the anti-LDL receptor antibody; the rates, however, were reduced to less than half by RAP. These suggest that the LDL receptor-related protein (LRP) or another LDL receptor family member(s) contributes to chylomicron remnant uptake and becomes the major mechanism of uptake when LDL receptors are absent. In contrast, the VLDL receptor was not involved as its absence did not affect chylomicron remnant metabolism. Similarly, the absence of apoE production did not affect the amount of remnant uptake; however, the proportion that was sensitive to RAP was eliminated. The level of LRP expression was not altered in these cells whereas there was a decrease in LDL receptors. This suggests that the apoE content of chylomicron remnants is sufficient for its recognition by LDL receptors but additional apoE is required for its uptake by the LRP and that there is an up-regulation of a non-LDL receptor family mechanism in apoE deficiency. Together these studies suggest that even in the absence of LDL receptors or apoE secretion, chylomicron remnants could contribute to lipid accumulation in the artery wall during atherogenesis.     

Secretion-capture role for apolipoprotein E in remnant lipoprotein metabolism involving cell surface heparan sulfate proteoglycans

To determine the impact of enhanced apolipoprotein (apo) E secretion on the mechanism of remnant lipoprotein uptake, rat hepatoma cells (McA-RH7777) were stably transfected with normal human apoE3 or receptor binding-defective apoE-Leiden. After a 2-h incubation, the human apoE secreted from the transfected hepatocytes was 10-12 times greater than the endogenous rat apoE. The apoE3-transfected cells bound and internalized rabbit beta-very low density lipoproteins (beta-VLDL) to a much greater degree than did apoE-Leiden-transfected cells and nontransfected cells. The apoE3-secreting cells displayed a 2-3.5-fold enhancement of cell-associated beta-VLDL compared to either the apoE-Leiden-transfected or nontransfected cells. Fluorescently labeled beta-VLDL were observed to concentrate within intracellular granules of the apoE3-transfected cells, presumably within endosomes and lysosomes. Furthermore, electron microscopy revealed that the apoE3-secreting cells displayed abundant beta-VLDL and chylomicron remnants on their cell surfaces and microvilli, in contrast to non-transfected or apoE-Leiden-secreting cells. Electron microscopy also revealed an abundance of chylomicron remnants within intracellular vesicles and multivesicular bodies of apoE3-transfected hepatocytes. Heparinase treatment (3 units/ml) completely abolished the increased association of beta-VLDL with apoE3-transfected cells but did not affect the limited association of beta-VLDL with apoE-Leiden-transfected or nontransfected cells. We established that the apoE3-enriched beta-VLDL were bound to cell surface heparan sulfate proteoglycans, as was the newly synthesized and secreted apoE3 (approximately 12% of the total secreted apoE3 was released by heparinase and suramin; 4% by heparin). In addition, reisolation of beta-VLDL by fast performance liquid chromatography after their incubation with exogenous apoE3, with medium from apoE3-secreting cells, or with the apoE3-secreting cells themselves revealed that the particles were enriched in apoE3 and displayed enhanced binding. These results suggest a secretion-capture role for apoE and indicate an important role for heparan sulfate proteoglycans on the cell surface for remnant lipoprotein metabolism.     

Human recombinant apolipoprotein E redirects lipopolysaccharide from Kupffer cells to liver parenchymal cells in rats In vivo

Chylomicrons have been shown to protect mice and rats against a lethal dose of lipopolysaccharide and may serve as a therapeutic means to protect against endotoxemia. However, the requisite of isolation from human lymph hampers pharmaceutical application. Recently, we developed recombinant chylomicrons from commercially available lipids and human recombinant apolipoprotein E. The current study explored the effectiveness of these apoE-enriched emulsions in redirecting LPS from Kupffer cells to liver parenchymal cells. Upon injection into rats, 125I-LPS rapidly and specifically associated with the liver (64.3+/-3.1% of the injected dose) and spleen (4.1+/-0.7%). The uptake of LPS by the spleen was four- to fivefold reduced upon incubation with the apoE-enriched emulsion or free apoE (P < 0.0001), but not with emulsion alone or Lipofundin. Within the liver, 125I-LPS mainly associated with Kupffer cells. The uptake by Kupffer cells was eight- to ninefold reduced by the apoE-enriched emulsion or apoE alone (P < 0.01), and a 19.6-fold increased uptake ratio by liver parenchymal cells over Kupffer cells was observed. The emulsion without apoE had no effect on the in vivo kinetics of LPS. LPS interacted selectively with the apoE moiety of the recombinant chylomicron. Emulsion-associated and free apoE bound approximately two molecules of LPS, possibly by its exposed hydrophilic domain involving arginine residues. We anticipate that the protecting effect of endogenous chylomicrons against LPS-induced endotoxemia may result from the apoE moiety and that human recombinant apoE may serve as a therapeuticum to protect against endotoxemia.     

Decreased interleukin-12 (IL-12) from activated cord versus adult peripheral blood mononuclear cells and upregulation of interferon-gamma, natural killer, and lymphokine-activated killer activity by IL-12 in cord blood mononuclear cells

Remnants of lipoproteins, intestinal chylomicrons, and very low density lipoprotein (VLDL), are rapidly cleared from plasma and enter hepatocytes. It has been suggested that remnant lipoproteins are initially captured in the space of Disse by heparan sulfate proteoglycans (HSPGs), and that their subsequent internalization into hepatocytes is mediated by members of the LDL-receptor gene family. Similarly to lipoprotein remnants, malaria sporozoites are removed from the blood circulation by the liver within minutes after injection by Anopheles mosquitoes. The sporozoite's surface is covered by the circumsporozoite protein (CS), and its region II-plus has been implicated in the binding of the parasites to glycosaminoglycan chains of hepatocyte HSPGs. Lactoferrin, a protein with antibacterial properties found in breast milk and neutrophil granules, is also rapidly cleared from the circulation by hepatocytes, and can inhibit the hepatic uptake of lipoprotein remnants. Here we provide evidence that sporozoites, lactoferrin, and remnant lipoproteins are cleared from the blood by similar mechanisms. CS, lactoferrin, and remnant lipoproteins compete in vitro and in vivo for binding sites on liver cells. The relevance of this binding event for sporozoite infectivity is highlighted by our demonstration that apoliprotein E-enriched beta-VLDI and lactoferrin inhibit sporozoite invasion of HepG2 cells. In addition, malaria sporozoites are less infective in LDL-receptor knockout (LDLR -/-) mice maintained on a high fat diet, as compared with littermates maintained on a normal diet. We conclude that the clearance of lipoprotein remnants and sporozoites from the blood is mediated by the same set of highly sulfated HSPGs on the hepatocyte plasma membrane.     

Effects of chylomicrons and chylomicron remnants on endothelium-dependent relaxation of rat aorta

The effects of chylomicrons and chylomicron remnants on endothelium-dependent relaxation of rat aorta were studied in vitro. Chylomicrons and chylomicron remnants were prepared in vivo. Aortic rings were incubated with the lipoproteins for 45 min before the vessels were constricted with phenylephrine and concentration relaxation response curves constructed to carbachol, ATP, A23187 and S-nitroso-N-acetylpenicillamine. Maximum % relaxations to carbachol were significantly reduced by both chylomicrons and chylomicron remnants but responses to ATP and S-nitroso-N-acetylpenicillamine were unaffected. In addition, chylomicrons significantly inhibited A23187-induced relaxation, causing an increase in the EC50 value. Chylomicron remnants cause selective inhibition of carbachol-induced relaxation suggesting an action at the receptor or G protein-coupled component of the receptor-mediated activation of the L-arginine-nitric oxide pathway. Chylomicrons appear to be less selective in their inhibition of the endothelium-dependent relaxation. This study demonstrates that lipoprotein particles of dietary origin may cause endothelial cell dysfunction.     

Hepatic lipase facilitates the selective uptake of cholesteryl esters from remnant lipoproteins in apoE-deficient mice

We have investigated the role of hepatic lipase (HL) in remnant lipoprotein metabolism independent of lipolysis by using recombinant adenovirus to express native and catalytically inactive HL (HL-145G) in apolipoprotein (apo)E-deficient mice characterized by increased plasma concentrations of apoB-48-containing remnants. In the absence of apoE, the mechanisms by which apoB-48-containing remnants are taken up by either low density lipoprotein (LDL)-receptor or LDL-receptor-related protein (LRP) remain unclear. Overexpression of either native or catalytically inactive HL in apoE-deficient mice led to similar reductions (P > 0.5) in the plasma concentrations of cholesterol (41% and 53%) and non high density lipoprotein (HDL)-cholesterol (41% and 56%) indicating that even in the absence of lipolysis, HL can partially compensate for the absence of apoE in this animal model. Although the clearance of [3H]cholesteryl ether from VLDL was significantly increased (approximately 2-fold; P < 0. 02) in mice expressing native or inactive HL compared to luciferase controls, the fractional catabolic rates (FCR) of [125I-labeled] apoB- very low density lipoprotein (VLDL) in all three groups of mice were similar (P > 0.4, all) indicating selective cholesterol uptake. Hepatic uptake of [3H]cholesteryl ether from VLDL was greater in mice expressing either native HL (87%) or inactive HL-145G (72%) compared to luciferase controls (56%). Our combined findings are consistent with a role for HL in mediating the selective uptake of cholesterol from remnant lipoproteins in apoE-deficient mice, independent of lipolysis. These studies support the concept that hepatic lipase (HL) may serve as a ligand that mediates the interaction between remnant lipoproteins and cell surface receptors and/or proteoglycans. We hypothesize that one of these pathways may involve the interaction of HL with cell surface receptors, such as scavenger receptor (SR)-BI, that mediate the selective uptake of cholesteryl esters.     

Interpersonal relations and suicide ideation in China

The binding and internalization of chylomicron remnants in rat hepatocytes originating from the periportal and perivenous zones was compared. The hepatocyte sub-populations were separated by centrifugal elutriation and incubated with 125I-labelled chylomicron remnants at 37 degrees C (to measure binding and internalization) or 4 degrees C (to measure initial binding). Periportal and perivenous cells bound and internalised similar amounts of remnants up to a concentration of about 25 micrograms remnant protein per assay, but at higher concentrations the periportal cells were able to internalise significantly more remnants. When excess unlabelled low density lipoprotein was added to the incubations, little effect on the kinetics of either binding or internalization of the remnants was observed. Lactoferrin, an inhibitor of uptake via the remnant receptor, also did not affect the initial binding of the remnants to either cell type, but decreased internalization to similar extents in both sub-populations. These results suggest that periportal hepatocytes have a greater capacity for the uptake of chylomicron remnants than perivenous cells, and that the remnant receptor plays a more important role than the low density lipoprotein receptor in both sub-populations. This acinar heterogeneity parallels that reported previously for cholesterol de novo synthesis, bile formation, lipid content and hepatic lipase secretion.     

VLDL receptor mediates the uptake of human chylomicron remnants in vitro

The VLDL receptor has been described as a new member of the LDL receptor supergene family that specifically binds VLDL in vitro via apolipoprotein E and lipoprotein lipase. Both apolipoprotein E and lipoprotein lipase are constituents of chylomicron remnants, another triglyceride-rich lipoprotein which has been proposed as a physiological ligand for the VLDL receptor. We used human chylomicron remnants to study their uptake into LDL, receptor-deficient Chinese hamster ovary cells overexpressing the human VLDL receptor. The uptake into these cells was compared to that into cells transfected with an empty transfection vector. Human chylomicron remnants were produced in vitro by hydrolysis with lipoprotein lipase, and were labeled with 125I. The uptake of these remnants into the cells overexpressing the VLDL receptor was found to be about 3-fold higher than the uptake into the control cells. The addition of a surplus of either apolipoprotein E or inactivated lipoprotein lipase to the remnants led to an increase in particle uptake. The chylomicron remnant uptake was inhibited by addition of the 39 kDa receptor associated protein These in vitro experiments strongly support the idea that the VLDL receptor is a physiological receptor for chylomicron remnants. The increase of receptor-mediated uptake induced by the addition of apoE or lipoprotein lipase underlines the role of these two proteins in this process.     

Fluorometric determination of total retinyl esters in triglyceride-rich lipoproteins

A time-consuming sample preparation and measuring procedure is required for the quantitation of retinyl palmitate by HPLC. We developed a fluorometric method for the determination of total retinyl esters in chylomicrons, chylomicron remnants, and VLDL. This method is precise, sensitive, rapid, simple, and particularly useful for large-scale studies of postprandial lipid metabolism. Because the turbidity of postprandial lipemic samples interferes with the fluorescence measurement, all samples were incubated for 10 min with a clearing buffer containing esterase and detergents. This buffer eliminates the turbidity and hydrolyzes all retinyl esters to retinol. The fluorescence signal (excitation wavelength, 330 nm; emission wavelength, 490 nm) was linear from 0.1 mg/L up to 4 mg/L retinyl palmitate, and the CVs were 3.6% within-run and 5.1% within-series. A first application studied postprandial lipoproteins, which were first separated by ultracentrifugation and then subjected to size exclusion chromatography. Fluorescence analysis revealed that the chylomicron density fraction contains large amounts of chylomicron remnants.     

Schizophrenia and genetics: a review and critique for the psychiatric nurse

OBJECTIVES: To study the role of the LDL receptor in the clearance of chylomicron remnants in humans. DESIGN: Chylomicron remnant clearance was studied in five untreated subjects with heterozygous familial hypercholesterolaemia (FH) and nine normolipidaemic controls, by oral retinyl palmitate-fat loading tests. Fasting plasma triglycerides (TG), which are important determinators of chylomicron and remnant clearance, were not significantly different between FH (1.76+/-0.32 mmol L(-1), mean+/-SEM) and controls (1.26+/-0.18 mmol L(-1). Chylomicrons (Sf > 1000) and their remnants (Sf < 1000) were separated by flotation and their clearance was estimated by calculating the area under the 24 h-retinyl palmitate curve (AUC-RP). The factors determining chylomicron and remnant clearance were studied by univariate and multiple regression analysis. RESULTS: Triglyceride clearance in plasma, Sf > 1000 fractions and Sf < 1000 fractions was not significantly different between FH subjects and controls. In subjects with heterozygous FH, chylomicron remnant clearance was two-fold delayed (AUC-RP, 49.39+/-11.61 h.mg L(-1) compared to controls (27.45+/-3.95 h.mg L(-1); P = 0.048). Moreover, 28.4% higher fasting plasma TG in FH resulted in 44.4% higher areas under the remnant-curves compared to controls. The clearance of chylomicron RP was associated to plasma apo E (beta = 0.73, P = 0.011), plasma LDL cholesterol (beta = 0.62, P = 0.018) and plasma TG (beta = 0.58, P = 0.029). The clearance of remnant RP was associated to the diagnosis (FH vs. non-FH), but not to the well-known determinants of remnant clearance like plasma TG. CONCLUSIONS: The clearance of chylomicrons and large remnants isolated in the Sf > fraction depends primarily on the apo B, E (LDL) receptor and to a lesser extent on plasma triglycerides. The clearance of smaller chylomicron remnants isolated in the Sf < 1000 depends to a large extent on the apo B, E (LDL) receptor.     

Transgenic mice expressing high plasma concentrations of human apolipoprotein B100 and lipoprotein(a)

The B apolipoproteins, apo-B48 and apo-B100, are key structural proteins in those classes of lipoproteins considered to be atherogenic [e.g., chylomicron remnants, beta-VLDL, LDL, oxidized LDL, and Lp(a)]. Here we describe the development of transgenic mice expressing high levels of human apo-B48 and apo-B100. A 79.5-kb human genomic DNA fragment containing the entire human apo-B gene was isolated from a P1 bacteriophage library and microinjected into fertilized mouse eggs. 16 transgenic founders expressing human apo-B were generated, and the animals with the highest expression had plasma apo-B100 levels nearly as high as those of normolipidemic humans (approximately 50 mg/dl). The human apo-B100 in transgenic mouse plasma was present largely in lipoproteins of the LDL class as shown by agarose gel electrophoresis, chromatography on a Superose 6 column, and density gradient ultracentrifugation. When the human apo-B transgenic founders were crossed with transgenic mice expressing human apo(a), the offspring that expressed both transgenes had high plasma levels of human Lp(a). Both the human apo-B and Lp(a) transgenic mice will be valuable resources for studying apo-B metabolism and the role of apo-B and Lp(a) in atherosclerosis.     

Protection against atherogenesis in mice mediated by human apolipoprotein A-IV

Apolipoproteins are protein constituents of plasma lipid transport particles. Human apolipoprotein A-IV (apoA-IV) was expressed in the liver of C57BL/6 mice and mice deficient in apoE, both of which are prone to atherosclerosis, to investigate whether apoA-IV protects against this disease. In transgenic C57BL/6 mice on an atherogenic diet, the serum concentration of high density lipoprotein (HDL) cholesterol increased by 35 percent, whereas the concentration of endogenous apoA-I decreased by 29 percent, relative to those in transgenic mice on a normal diet. Expression of human apoA-IV in apoE-deficient mice on a normal diet resulted in an even more severe atherogenic lipoprotein profile, without affecting the concentration of HDL cholesterol, than that in nontransgenic apoE-deficient mice. However, transgenic mice of both backgrounds showed a substantial reduction in the size of atherosclerotic lesions. Thus, apoA-IV appears to protect against atherosclerosis by a mechanism that does not involve an increase in HDL cholesterol concentration.     

Resistance of chylomicron and VLDL remnants to post-heparin lipolysis in ApoE-deficient mice: the role of apoE in lipoprotein lipase-mediated lipolysis in vivo and in vitro

The interaction of lipoprotein lipase (LPL) with triglyceride-rich lipoproteins is governed by a number of factors, such as apolipoprotein (apo) C-II. The role of apoE in lipolysis is controversial. We made the unexpected observation that apoE-deficient mice were resistant to heparin-induced lipolysis; this study aims at examining the underlying mechanism for this observation. Compared to wild-type mice, apoE-deficient mice had significantly higher very low density lipoprotein (VLDL) and chylomicron remnant (VLDL/CMR) concentrations and moderately lower lipase activity (15.5 +/- 1.3 mU/ml vs. 22.9 +/- 2.5 mU/ml). Unlike in wild-type mice where the injection of heparin reduced total plasma triglycerides by 50% and VLDL/CMR triglycerides by over 95%, the injection of heparin into apoE-deficient mice did not significantly affect plasma lipids. Similarly, in vitro, purified human LPL (hLPL) almost completely hydrolyzed VLDL/CMR isolated from wild-type mice, but had no effect on VLDL/CMR from apoE-deficient mice. However, when the amount of apoE-deficient VLDL/CMR was reduced to an equivalent level as in wild-type mice, LPL hydrolyzed 94% of VLDL/CMR triglycerides. In order to increase the ratio of LPL to VLDL/CMR in vivo, we injected an adenovirus containing the human LPL cDNA into apoE-deficient mice, which produced marked liver-specific overexpression of LPL and significant reduction of VLDL/CMR (93%) and total plasma triglyceride concentrations (87%). Thus, apoE is not required for LPL activity in vivo or in vitro. Under certain pathological conditions, such as severe hyperlipidemia, the LPL pathway may be saturated and efficient lipolysis can proceed only if the ratio of substrate particles to LPL is adjusted to a more normal range.     

Hepatic apo E expression is required for remnant lipoprotein clearance in the absence of the low density lipoprotein receptor

According to the secretion-capture model of remnant lipoprotein clearance, apo E secreted by hepatocytes into the space of Disse serves to enrich the remnants with a ligand for receptor-mediated lipoprotein endocytosis. Current evidence supports a two-receptor model of lipoprotein removal, in which apo E-containing remnants bind either the low density lipoprotein receptor (LDLR) or the LDLR-related protein (LRP). Recently, we demonstrated that reconstitution of apo E(-/-) mice with apo E(+/+) marrow results in normalization of plasma lipoprotein levels, indicating that hepatic expression of apo E is not required for remnant clearance and calling into question the relevance of the secretion-capture mechanism. To dissect the relative contributions of LDLR and LRP to the cellular catabolism of remnant lipoproteins by the hepatocyte, bone marrow transplantation (BMT) was used to reconstitute macrophage expression of apo E in mice that were null for expression of both apo E and the LDLR. Reconstitution of macrophage apo E in apo E(-/-)/LDLR(-/-) mice had no effect on serum lipid and lipoprotein concentrations, although it produced plasma apo E levels up to 16-fold higher than in C57BL/6 controls. Immunocytochemistry of hepatic sections revealed abundant staining for apo E in the space of Disse, but no evidence of receptor-mediated endocytosis of remnant lipoproteins. Transient expression of human LDLR in the livers of apo E(+/+)--> apo E(-/-)/LDLR(-/-) mice by adenoviral gene transfer resulted in normalization of serum lipid levels and in the clearance of apo E-containing lipoproteins from the space of Disse. We conclude that whereas the LDLR efficiently clears remnant lipoproteins irrespective of the site of origin of apo E, endocytosis by the chylomicron remnant receptor (LRP) is absolutely dependent on hepatic expression of apo E. These data demonstrate in vivo the physiologic relevance of the apo E secretion-capture mechanism in the liver.     

Accumulation of large very low density lipoprotein in plasma during intravenous infusion of a chylomicron-like triglyceride emulsion reflects competition for a common lipolytic pathway

Very low density lipoproteins (VLDL) are produced in the liver and contain apolipoprotein (apo) B-100 and endogenous lipids. By contrast, ingestion of fat leads to formation of chylomicrons containing apoB-48 secreted from the intestine. In this study, a 60-min intravenous infusion of a chylomicron-like triglyceride emulsion was given to healthy young men to examine whether competition between chylomicrons and VLDL for the same lipolytic pathway explains the increase in VLDL seen after meals. The responses of two major VLDL subfractions were determined by measuring the concentrations of apoB-100 in fractions of triglyceride-rich lipoproteins with Svedberg flotation rates of 60-400 (large VLDL) and 20-60 (small VLDL) that were separated from plasma by density gradient ultracentrifugation. A threefold elevation in plasma triglycerides was observed during the infusion together with a consistent linear increase of large VLDL. The rate at which large VLDL accumulated in plasma differed markedly among individuals and was not enhanced by doubling of the infusion rate. The response of small VLDL was more heterogeneous; however, a decrease was seen in most subjects. The combined pattern for the two VLDL species is what would be expected if large VLDL particles are the precursors of smaller VLDL species and if lipolysis of large VLDL is inhibited through competition from the triglyceride emulsion. The extent to which the triglyceride emulsion inhibited the lipolysis of VLDL and/or influenced the synthesis rate of large VLDL was estimated from simultaneous stable isotope studies. The emulsion caused a 75-90% block of the conversion of large VLDL apoB to small VLDL apoB and there was no sign of enhanced synthesis of large VLDL after infusion of the triglyceride emulsion. The corollary of these findings is that chylomicrons and their remnants impede the normal lipolytic degradation of VLDL and could thereby be indirectly implicated in the generation of atherogenic remnant lipoproteins.     

The influence of dietary protein intake on milk production and blood composition of high-yielding dairy cows

Isolated rat adipocytes were incubated with serum lipoproteins or lymphchylomicrons which contained 14c-labeled cholesterol. The specific activity of lipoprotein free cholesterol decreased and that of cellular free cholesterol increased linearly up to 7 h. At this time the cell cholesterol specific activity was only 11% of that of medium cholesterol indicating that the rate of exchange was slow. The specific activity of lipoprotein esterified cholesterol remained unchanged while that of cells showed a slight increase suggesting esterification of incorporated free cholesterol. No detectable change of the lipoprotein or cellular cholesterol concentration occurred indicating that the uptake of radioactive free cholesterol was due to exchange without net movement of sterol. The radioactive cholesterol was incorporated into both membrane fraction and the fat droplet of the adipocytes. The rate of cholesterol exchange was temperature-dependent but it was not influenced by the metabolic state of the cells and not by addition of metabolic inhibitors. Trypsin or pronase treatment of the cells were without influence on the rate of the exchange and denaturation of the plasma lipoproteins with formalin increased the rate of exchange. These results indicate that the exchange of cholesterol is a physical chemical process, which is not linked to energy metabolism of the cells, and which is not mediated by either specific lipoprotein receptors on fat cell membranes or pinocytic uptake of lipoproteins. The rate of free cholesterol exchange showed a linear correlation with the concentration of lipoprotein particles in the medium. The relative transfer rate was highest for chylomicrons and decreased in order chylomicron remnants greater than very low density lipoprotein greater than low density lipoprotein greater than high density lipoprotein. A saturation of the system could be obtained only with high density lipoprotein.