Concentration and distribution of apolipoproteins A-I and E in normolipidemic, WHHL and diet-induced hyperlipidemic rabbit sera.

Two sandwich-type enzyme immunoassays have been developed to measure apolipoproteins A-I and E in rabbit serum. Specific goat antibodies were purified by affinity chromatography and used both for coating and for preparing antibody-peroxydase conjugates. The sensitivity of these assays is sufficient to allow studies of apo A-I and E distribution in lipoproteins fractionated by gel filtration from 50 microliters of serum. In WHHL rabbits, apo A-I is 5-fold lower (5.2 +/- 2.5 mg/dl) and apo E is 8-fold higher (9.9 +/- 3.5 mg/dl) than in normolipidemic rabbits (29 +/- 4.3 mg/dl and 1.3 +/- 0.5 mg/dl, respectively). In hyperlipidemic rabbits, fed 2 months on a 0.5% cholesterol diet, the apo A-I level was similar (32 +/- 12 mg/dl) to that of normolipidemic rabbits, but the apo E level is 12-fold higher (15.1 +/- 5.5 mg/dl). In addition, HDL particles were enriched with cholesterol and apo E. The bulk of apo E and cholesterol is located in large beta-VLDL in diet-induced hyperlipidemia, whereas they are mainly located in smaller size beta-VLDL in WHHL rabbits. In normolipidemic rabbits apo E occurs mainly in HDL, and cholesterol is distributed in the main three lipoprotein fractions VLDL, LDL and HDL. Interestingly, HDL of WHHL rabbit are deficient in apo A-I. These results are compatible with profound perturbations of lipoprotein composition and metabolism in atherogenic hyperlipidemia.     

Characterization of monoclonal anti-rabbit apolipoprotein E antibodies and chemical composition of lipoproteins separated by anti-apolipoprotein E immuno-affinity chromatography

Six mouse monoclonal antibodies against rabbit apolipoprotein E (apo E) have been developed. Of these monoclonal antibodies, clone 5 revealed a high affinity for purified apo E, very low density lipoprotein (VLDL) and beta-VLDL. This monoclonal antibody was used to prepare an immunoaffinity column. Coupled to Sepharose 4B, this antibody allowed complete removal of lipoproteins containing apo E from plasma of New Zealand white (NZW) rabbits; 62, 46, 14, and 3% of VLDL-, IDL-, LDL-, and HDL-protein, respectively, were bound to the anti-apo E affinity column. The bound VLDL was significantly rich in free cholesterol (FC) and cholesteryl esters (CE) relative to the unbound VLDL, whereas bound IDL, LDL and HDL were significantly rich in FC only. All of the bound fractions were characterized by significantly increased ratios of FC/phospholipids (PL). These results indicate that the two lipoprotein populations with and without apo E have different lipid compositions. The relatively high content of cholesterol in lipoproteins containing apo E suggests a contribution of apo E to plasma cholesterol transport.     

Mechanisms of inhibition by apolipoprotein C of apolipoprotein E-dependent cellular metabolism of human triglyceride-rich lipoproteins through the low density lipoprotein receptor pathway

The mechanism of inhibition by apolipoprotein C of the uptake and degradation of triglyceride-rich lipoproteins from human plasma via the low density lipoprotein (LDL) receptor pathway was investigated in cultured human skin fibroblasts. Very low density lipoprotein (VLDL) density subfractions and intermediate density lipoprotein (IDL) with or without added exogenous recombinant apolipoprotein E-3 were used. Total and individual (C-I, C-II, C-III-1, and C-III-2) apoC molecules effectively inhibited apoE-3-mediated cell metabolism of the lipoproteins through the LDL receptor, with apoC-I being most effective. When the incubation was carried out with different amounts of exogenous apoE-3 and exogenous apoC, it was shown that the ratio of apoE-3 to apoC determined the uptake and degradation of VLDL. Excess apoE-3 overcame, at least in part, the inhibition by apoC. ApoC, in contrast, did not affect LDL metabolism. Neither apoA-I nor apoA-II, two apoproteins that do not readily associate with VLDL, had any effect on VLDL cell metabolism. The inhibition of VLDL and IDL metabolism cannot be fully explained by interference of association of exogenous apoE-3 with or displacement of endogenous apoE from the lipoproteins. IDL is a lipoprotein that contains both apoB-100 and apoE. By using monoclonal antibodies 4G3 and 1D7, which specifically block cell interaction by apoB-100 and apoE, respectively, it was possible to assess the effects of apoC on either apoprotein. ApoC dramatically depressed the interaction of IDL with the fibroblast receptor through apoE, but had only a moderate effect on apoB-100. The study thus demonstrates that apoC inhibits predominantly the apoE-3-dependent interaction of triglyceride-rich lipoproteins with the LDL receptor in cultured fibroblasts and that the mechanism of inhibition reflects association of apoC with the lipoproteins and specific concentration-dependent effects on apoE-3 at the lipoprotein surface.     

Alterations in plasma lipoproteins and apolipoproteins associated with estrogen-induced hyperlipidemia in the laying hen

The laying hen represents a physiological model in which the mechanisms of action of estrogens on lipid transport can be evaluated. The plasma lipoproteins in the laying hen were subfractionated into discrete particle species by isopycnic density gradient ultracentrifugation and the physicochemical properties and apolipoprotein contents of individual subfractions evaluated. The qualitative and quantitative aspects of this estrogen-specific profile were then compared to those of the immature chicken. As observed earlier, estrogens induced dramatic elevation in very-low-density lipoproteins (VLDL) (up to 900 mg/dl). Indeed, triglyceride-rich lipoproteins with densities up to 1.035 g/ml, i.e. VLDL and their remnants, behaved as a continuum which displayed little variation in size (20.5-21 nm), electrophoretic mobility (beta-like) and apolipoprotein content; apo B-100 (540 kDa) predominated while apo A-I (27 kDa), apo VLDL-II (19 kDa) and an apo-C-like protein (13 kDa) were present as minor components. The typical high-density lipoproteins (HDL) in the immature chicken were replaced by a lipoprotein population whose physicochemical properties were quite distinct. Thus these particles were distributed as a single, asymmetric peak over the density range 1.030-1.158 g/ml, a wide interval which overlapped that of apo-B-rich particles at its lower limit. The rho 1.030-1.158 g/ml lipoproteins were present at concentrations (approximately equal to 200 mg/dl) some twofold to threefold lower than those of HDL in immature birds. Furthermore, they displayed physical and chemical properties in common with both low-density lipoproteins (LDL) and HDL and were LDL-like in exhibiting beta mobility but HDL-like in size (9-15 nm diameter). Their protein moiety was also HDL-like in its predominant content of apo A-I; small amounts of apo VLDL-II and the apo-C-like protein were also detected. Substantial amounts of lipid were found at rho greater than 1.195 g/ml: such substances are absent in the immature chicken and may reflect the presence of vitellogenins. The hyperestrogenic state in the laying hen is therefore associated with major modifications in lipoprotein and apolipoprotein profile. Such modifications may be of relevance to clinical disorders involving estrogen-induced hyperlipidemia.     

A comparative study of serum lipoproteins in rabbits fed a natural ingredient diet or low-fat, cholesterol-free, semipurified diets containing casein or isolated soy protein

In rabbits fed a cholesterol-free, semipurified diet containing isolated soy protein, the average total serum cholesterol level was similar to that of rabbits fed a natural ingredient (chow) diet. However, the cholesterol and protein levels in very low density (VLDL) and low density lipoproteins (LDL) tended to increase, while the levels in high density lipoproteins (HDL) were reduced to about half of those on the chow diet, with little change in the cholesterol to protein ratio. Substitution of casein for soy protein in the semipurified diet caused a four- to five-fold increase in total serum cholesterol and a doubling of lipoprotein protein, with an increase of 1.4- to 3.0-fold in the cholesterol to protein ratio of the different lipoprotein fractions. Analysis of the apoproteins (apo) of the plasma lipoproteins indicated that apo B, E, and C all tended to increase in the VLDL and LDL of rabbits fed the soy protein diet compared with those fed chow diet. The levels of each of the apoproteins were increased further by substituting casein for soy protein in the semipurified diet. In this case, apo E showed the greatest relative increase (2.7-fold) in VLDL, while apo B and E were increased to a similar extent (about 4-fold) in LDL. Apo C was approximately doubled in each of these fractions. The apo A content in HDL of rabbits fed the semipurified diets was about half that of rabbits fed chow diet. No marked changes were noted in the apo E or C content of HDL. Separation of isoforms of the soluble apoproteins showed variations between individual animals, but these variations seemed largely unrelated to diet. The results of these studies indicate that semipurified diets produce changes in the serum lipoprotein patterns of rabbits that are only partly due to the protein component of these diets.     

Lipoprotein metabolism in the suckling rat: characterization of plasma and lymphatic lipoproteins

Suckling rat plasma contains (in mg/dl): chylomicrons (85 +/- 12); VLDL (50 +/- 6); LDL (200 +/- 23); HDL1 (125 +/- 20); and HDL2 (220 +/- 10), while lymph contains (in mg/dl): chylomicrons (9650 +/- 850) and VLDL (4570 +/- 435) and smaller amounts of LDL and HDL. The lipid composition of plasma and lymph lipoproteins are similar to those reported for adults, except that LDL and HDL1 have a somewhat higher lipid content. The apoprotein compositions of plasma lipoproteins are similar to those of adult lipoproteins except for the LDL fraction, which contains appreciable quantities of apoproteins other than apoB. Although the LDL fraction was homogeneous by analytical ultracentrifugation and electrophoresis, the apoprotein composition suggests the presence of another class of lipoproteins, perhaps a lipid-rich HDL1. The lipoproteins of lymph showed low levels of apoproteins E and C. The triacylglycerols in chylomicrons and VLDL of both lymph and plasma are rich in medium-chain-length fatty acids, whereas those in LDL and HDL have little or none. Phospholipids in all lipoproteins lack medium-chain-length fatty acids. The cholesteryl esters of the high density lipoproteins are enriched in arachidonic acid, whereas those in chylomicrons, VLDL, and LDL are enriched in linoleic acid, suggesting little or no exchange of cholesteryl esters between these classes of lipoproteins. The fatty acid composition of phosphatidylcholine, sphingomyelin, and lysophosphatidylcholine were relatively constant in all lipoprotein fractions, suggesting ready exchange of these phospholipids. However, the fatty acid composition of phosphatidylethanolamine in plasma chylomicrons and VLDL differed from that in plasma LDL, HDL1, and HDL2. LDL, HDL1, and HDL2 were characterized by analytical ultracentrifugation and shown to have properties similar to that reported for adult lipoproteins. The much higher concentration of triacylglycerol-rich lipoproteins in lymph, compared to plasma, suggests rapid clearance of these lipoproteins from the circulation.     

Metabolism of apolipoprotein E-containing human plasma lipoproteins by rat and human cells in culture.

Cultured preadipocytes from rat epididymal fat pads were able to bind, internalize, and degrade human plasma very-low-density lipoproteins (VLDL) more efficiently than low-density lipoproteins (LDL). VLDL, but not LDL, activated acyl-CoA: cholesterol acyltransferase (ACAT) and increased cholesterol accumulation in these cells. However, trypsin-treated VLDL (T-VLDL) lost the capacity to bind, activate ACAT, and increase cholesterol accumulation. After the treatment of VLDL with trypsin, SDS/polyacrylamide-gel electrophoresis and immunoblotting showed that apolipoprotein E (apo E) was completely degraded, whereas apolipoprotein CII (apo C-II) was preserved. ApoE complexed with dimyristoyl phosphatidylcholine (DMPC) was able to complete with VLDL for binding to the cells. Although T-VLDL did not bind to the preadipocytes, these cells accumulate triacylglycerols from T-VLDL, presumably after lipolysis, as efficiently as from native VLDL. Rat smooth muscle cells and skin fibroblasts also bind and metabolize human VLDL better than LDL. However, human skin fibroblasts and omental preadipocytes metabolized LDL better than VLDL. These studies indicate that rat tissues can recognize and metabolize apoE-containing human plasma VLDL although they cannot recognize human LDL.     

Species differences of macrophage very low-density-lipoprotein (VLDL) receptor protein expression

Triglyceride-rich lipoproteins (TGRLs) and low-density-lipoprotein (LDL) cholesterol are independent risk factors for coronary artery disease. We have previously proposed that the very low-density-lipoprotein (VLDL) receptor is one of the receptors required for foam cell formation by TGRLs in human macrophages. However, the VLDL receptor proteins have not been detected in atherosclerotic lesions of several animal models. Here we showed no VLDL receptor protein was detected in mouse macrophage cell lines (Raw264.7 and J774.2) or in mouse peritoneal macrophages in vitro. Furthermore, no VLDL receptor protein was detected in macrophages in atherosclerotic lesions of chow-fed apolipoprotein E-deficient or cholesterol-fed LDL receptor-deficient mice in vivo. In contrast, macrophage VLDL receptor protein was clearly detected in human macrophages in vitro and in atherosclerotic lesions in myocardial infarction-prone Watanabe-heritable hyperlipidemic (WHHLMI) rabbits in vivo. There are species differences in the localization of VLDL receptor protein in vitro and in vivo. Since VLDL receptor is expressed on macrophages in atheromatous plaques of both rabbit and human but not in mouse models, the mechanisms of atherogenesis and/or growth of atherosclerotic lesions in mouse models may be partly different from those of humans and rabbits.     

The disappearance rate of human versus rat intermediate density lipoproteins from rat liver perfusion.

The aim of this work was to compare the disappearance rate of human and rat intermediate density lipoproteins (IDL) using the rat liver perfusion system. Human and rat IDL were produced in vitro by incubating human or rat very low density lipoproteins (VLDL) with either rat post-heparin plasma (method I) or a resolubilized isopropanol precipitate of rat post-heparin plasma (method II). With both methods, the degree of triacylglycerol lipolysis was approximately 55%. The different preparations of IDL were labelled with 125I and added to perfusates of rat livers. The disappearance rates of 125I-labelled IDL were monitored by measuring the radioactivity associated with apolipoprotein (apo) B in the perfusate during a 15-min period. Both human and rat IDL prepared with method I had an increased apoE to apoC ratio as compared with their native counterparts. Furthermore, human IDL had a significantly higher apoE to apoC ratio than rat IDL. However, when IDL were produced in the absence of exchangeable apolipoproteins (method II), no change in the apoE to apoC ratios was observed for the transformation of VLDL to IDL and the ratios were similar for human and rat IDL. Despite these differences, human IDL were always removed at a lower rate than rat IDL. The only striking difference between the two types of IDL made by method II was that the apoB100 to apoB48 ratio was considerably higher in human than in rat IDL. These results suggest that the apoB100 to apoB48 ratio is likely to be responsible for the observed differences in liver uptake between rat and human IDL.     

Assembly of lipoprotein (a) in transgenic rabbits expressing human apolipoprotein (a)

The study of human lipoprotein (a) [Lp(a)] has been hampered due to the lack of appropriate animal models since apolipoprotein (a) [apo(a)] is found only in primates and humans. In addition, human apo(a) in transgenic mice can not bind to murine apoB to form Lp(a) particles. In this study, we generated three independent transgenic rabbits expressing human apo(a) in their plasma at 1.8-4.5 mg/dl. In the plasma of transgenic rabbits, unlike the plasma of transgenic mice, about 80% of the apo(a) was covalently associated with rabbit apo-B and was contained in the fractions with density 1.02-1.10 g/ml, indicating the formation of Lp(a). These results suggest that transgenic rabbits expressing human apo(a) exhibit efficient assembly of Lp(a) and can be used as an animal model for the study of human Lp(a).     

Reduction in VLDL, but not HDL, in plasma of rats deficient in choline

We have analyzed plasma lipoprotein levels in young male rats fed a choline-deficient diet for 3 days. We confirmed previous studies that choline deficiency promotes 6.5-fold accumulation of triacyglycerol in the liver (23.9 +/- 6.0 versus 3.69 +/- 0.92 mumol/g liver) and reduction of triacylglycerol concentration in plasma by 60% (0.17 +/- 0.04 versus 0.46 +/- 0.10 mumol/mL plasma). Agarose gel electrophoresis showed that the plasma very low density lipoprotein (VLDL) levels were reduced in choline-deficient rats, but the concentration of plasma high density lipoproteins (HDL) was not affected. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis of fractionated plasma lipoproteins revealed that the concentrations of apolipoproteins (apo) BH, BL, and E in VLDL from choline-deficient rats were 37.1, 11.0, and 37.2% of normal levels, respectively. In contrast, the amount of apo A-I, the major one in HDL, was almost unchanged. Correspondingly, there were decreased lipid (mainly phosphatidylcholine and triacylglycerol) levels in VLDL from choline-deficient rats, but no change in the levels of phosphatidylcholine, cholesterol, and cholesterol ester in HDL. There were similar levels of apo B and E (components of VLDL) in homogenates of livers from normal and choline-deficient rats, as determined by immunoblotting. These results support the hypothesis that choline deficiency causes reduction of VLDL, but not HDL, levels in plasma as a consequence of impaired hepatic VLDL secretion.     

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.     

Distribution and characterization of the serum lipoproteins and apoproteins in the mouse, Mus musculus

Murine lipoproteins were separated into nine subfractions by a density gradient ultracentrifugal procedure. They were characterized by electrophoretic, immunological, chemical, and morphological analyses, and their protein moieties were defined according to charge, molecular weight, and isoelectric point. HDL predominated (approximately 500 mg/dl serum), the mode of its distribution being situated in the d 1.09-1.10 g/ml (F 1.21 approximately 4) region. Chemical analysis showed subfractions of d 1.085-1.136 g/ml to resemble human HDL3 closely, including the presence of apoA-I (Mr 25,000-27,000) as their major apolipoprotein. An apoA-II-like protein, of Mr 8400 (in monomeric form), was also tentatively identified. In electrophoretic mobility and chemical composition, the d 1.060-1.085 g/ml subfraction (approximately 10% of total HDL) was distinct and akin to human HDL2. ApoA-I represented approximately 60% of its complement of low molecular weight apoproteins. The density range used for separation of human HDL2 (d 1.066-1.100 g/ml) by gradient ultracentrifugation is inadequate in the mouse, and the d 1.060-1.085 g/ml interval is more appropriate. The 1.063 g/ml boundary for separation of mouse LDL from HDL was unsuitable. Immunological and electrophoretic studies revealed that alpha-migrating lipoproteins were present in the d 1.046-1.060 g/ml range, a finding consistent with their enrichment in apoA-I; apoE-, apoA-II-, and apoC-like proteins were also detected. These findings indicate the presence of HDL1 particles. Murine apoA-I and apoB-like proteins of higher (apoBH) and lower (apoBL) molecular weight were constituents of the d 1.033-1.046 g/ml fraction. Alternative techniques, such as electrophoresis in starch block, are therefore a prequisite for separation of apoB from alpha-migrating, apoA-I-containing lipoproteins in the low density range in mouse serum. The LDL class (d 1.023-1.060 g/ml) amounted to only approximately 20% of the total murine lipoproteins of d less than 1.188 g/ml (65-70 mg/dl serum). Particles were richer In triglyceride, larger in diameter (mean 244 A), and more heterogeneous than typical of man. VLDL (40-80 mg/dl serum) was triglyceride-rich (66% by weight) and similarly heterogeneous in size (mean diameter 494 A; range 270-750 A). ApoBH and apoBL were prominent in murine VLDL, and cross-reacted with an antiserum to human apoB. ApoE- and apoA-I-like proteins were also detectable in apoVLDL, as was a protein of 70,000-75,000 mol wt. The presence of murine apolipoproteins analogous to human apoB and apoE was confirmed by the immunological cross-reactivities of VLDL and LDL with monospecific antisera to the human proteins. The marked similarity of lipoprotein and apolipoprotein profile in the mouse and rat is notable. Since murine VLDL contains apoE and apoBL, this resemblance may extend to the metabolism of chylomicron remnants and hepatic VLDL in the two species.     

Synthesis and secretion of lipoproteins by human hepatocytes in culture

Summary Confluent monolayers of normal human hepatocytes obtained by collagenase perfusion of liver pragments were incubated in a serum-free medium. Intracellular apolipoproteins apo AI, apo C, apo B, and apo E were detected between Day 1 and Day 6 of the culture by immunoenzymatic staining using polyclonal antibodies directed against these apoproteins and monoclonal antibodies directed against both forms of apo B (B100 and B48). Translation of mRNA isolated from these hepatocytes in an acellular system revealed that apo AI and apo E were synthesized as the precusor forms of mature plasma apo AI and apo E. Three lipoprotein fractions corresponding to the density of very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL) were isolated from the medium at Day 5 of culture and examined by electron microscopy after negative staining. VLDL and LDL particles are similar in size and shape to plasma lipoproteins; spherical HDL are larger than normal plasma particles isolated at the same density. Their protein represented 44, 19.5, and 36.5% respectively, of the total lipoprotein protein. The secretion rate of VLDL protein corresponded to that measured in primary cultures of rat hepatocytes. After incorporation of [³H]glycerol, more than 92% of the [³H]triglyceride secreted into the medium was recovered in the VLDL fraction. These results demonstrate that primary cultures of normal human hepatocytes are able to synthesize and secrete lipoproteins and thus could be a useful model to study lipoprotein metabolism in human liver.     

Effect of fasting on the composition of plasma lipoproteins in cholesterol-fed diabetic rabbits

Cholesterol-fat feeding is associated with unusual alterations in the composition of plasma lipoproteins in alloxan-diabetic rabbits. In the present study plasma lipoprotein lipid and apoprotein composition was studied before and after 48 hr of fasting in cholesterol-fed diabetic and control rabbits in order to further characterize these alterations. Compared with control rabbits, the diabetic rabbits had similar plasma cholesterol levels, but 100-fold higher triglyceride levels prior to fasting. These plasma lipids were distributed mainly to large, Sf greater than 400 plasma lipoproteins in the diabetic rabbits, and to beta-VLDL in control rabbits. Sf greater than 400 lipoproteins, VLDL, IDL, LDL, and HDL from diabetic rabbits had triglyceride as the predominant lipoprotein core lipid. Sf greater than 400 lipoproteins and VLDL from diabetic rabbits had lesser amount of apoprotein E, and greater amounts of apoproteins A-I, A-IV, and B-48 as percent of total apoprotein mass in comparison with control rabbits. Fasting reduced plasma triglyceride levels by 55% in diabetic rabbits. Sf greater than 400 lipoprotein and VLDL triglyceride content decreased but remained a major core lipid. Fasting eliminated apoproteins A-I and A-IV from Sf greater than 400 lipoproteins and VLDL, but had no significant effect on apoB-48 content. Insulin treatment of the diabetic rabbits reduced plasma triglyceride by approximately 90% resulting in cholesteryl ester-rich particles reassembling beta-VLDL both in the Sf greater than 400 lipoprotein and VLDL fractions. These results indicate that the alterations in plasma lipoproteins in cholesterol-fed diabetic rabbits result from the presence in the d less than 1.006 g/ml plasma lipoprotein class of partially metabolized, intestinally derived particles.     

Kinetics and mechanism of exchange of apolipoprotein C-III molecules from very low density lipoprotein particles

Transfer of apolipoprotein (apo) molecules between lipoprotein particles is an important factor in modulating the metabolism of the particles. Although the phenomenon is well established, the kinetics and molecular mechanism of passive apo exchange/transfer have not been defined in detail. In this study, the kinetic parameters governing the movement of radiolabeled apoC molecules from human very low density lipoprotein (VLDL) to high density lipoprotein (HDL3) particles were measured using a manganese phosphate precipitation assay to rapidly separate the two types of lipoprotein particles. In the case of VLDL labeled with human [14C]apoCIII1, a large fraction of the apoCIII1 transfers to HDL3 within 1 minute of mixing the two lipoproteins at either 4 degrees or 37 degrees C. As the diameter of the VLDL donor particles is decreased from 42-59 to 23-25 nm, the size of this rapidly transferring apoCIII1 pool increases from about 50% to 85%. There is also a pool of apoCIII1 existing on the donor VLDL particles that transfers more slowly. This slow transfer follows a monoexponential rate equation; for 35-40 nm donor VLDL particles the pool size is approximately 20% and the t1/2 is approximately 3 h. The flux of apoCIII molecules between VLDL and HDL3 is bidirectional and all of the apoCIII seems to be available for exchange so that equilibrium is attained. It is likely that the two kinetic pools of apoCIII are related to conformational variations of individual apo molecules on the surface of VLDL particles. The rate of slow transfer of apoCIII1 from donor VLDL (35-40 nm) to acceptor HDL3 is unaffected by an increase in the acceptor to donor ratio, indicating that the transfer is not dependent on collisions between donor and acceptor particles. Consistent with this, apoCIII1 molecules can transfer from donor VLDL to acceptor HDL3 particles across a 50 kDa molecular mass cutoff semipermeable membrane separating the lipoprotein particles. These results indicate that apoC molecules transfer between VLDL and HDL3 particles by an aqueous diffusion mechanism.     

Isolation and characterization of lipoprotein of density less than 1.006 g/ml from rat hepatic lymph

The lipoprotein composition of rat hepatic lymph was studied using an animal model. The hepatic lymph duct of the adult male rat was cannulated and the hepatic lymph was collected. Hepatic lymph contains less than 1% of the total triglyceride output from the liver. Agarose gel electrophoresis of hepatic lymph showed the presence of two major lipoproteins bands, the alpha-migrating HDL and a band moving between plasma beta and pre-beta bands. Lipoprotein of density p less than 1.006 g/ml was then isolated by ultracentrifugation and it was found to correspond to the slow-moving pre-beta band. There was no difference in the mean diameter of hepatic lymph VLDL (64.4 nm) and that of plasma VLDL (64.6 nm). Compared with plasma VLDL, hepatic lymph VLDL has significantly more phospholipid (40% by weight), a higher cholesterol/cholesteryl ester ratio, and a marked reduction in triglyceride content (40% by weight). Although both plasma VLDL and hepatic lymph VLDL have apoE and apoB as the major apolipoproteins, there are other marked differences in apolipoprotein composition. Hepatic lymph VLDL has significantly less apoC and the apoB of hepatic lymph VLDL is predominantly the apoB240k (mol wt 240,000), with a small amount of the apoB330k (mol wt 335,000). On the other hand, plasma VLDL has an equal proportion of both apoB240k and apoB330k. This study presents for the first time the lipid and protein composition of rat hepatic lymph VLDL. Furthermore it has provided evidence that the hepatic lymph duct-cannulated rat can be used as an in vivo model for studying the secretion of nascent hepatic lymph VLDL.     

The enhanced cellular uptake of very-low-density lipoprotein enriched in apolipoprotein E.

We have recently reported an increased clearance of plasma very-low-density lipoprotein (VLDL) after intravenous injection of apolipoprotein (apo) E in Watanabe heritable hyperlipidemic (WHHL) rabbits. In the present study, we have investigated the cellular uptake of VLDL enriched in apo E (VLDL-E) which had been incubated with purified rabbit apo E. VLDL-E was taken up approx. 2-fold more than VLDL in human skin fibroblast, human monocyte-derived macrophage and Hep G2 cell and its degradation was least in macrophage. To characterize the binding of VLDL-E, we performed a binding assay using hepatic endosome isolated from estradiol-treated rats and we observed both increased EDTA-sensitive and -resistant binding of VLDL-E on endosome. Ligand blotting of hepatic endosome demonstrated two major bands of LDL receptor (130 and 260 kDa protein) and a minor band of LDL receptor-related protein (580 kDa protein) with a ligand of VLDL-E. These results suggested that VLDL-E was endocytosed in liver through a similar pathway among three cell types, and enrichment of apo E in VLDL enhanced the uptake of VLDL not only via an EDTA-sensitive binding site (classical LDL receptor) but also via other binding sites including an EDTA-resistant binding site and an LDL receptor-related protein.     

Acute inhibition of hepatic lipase and increase in plasma lipoproteins after alcohol intake

Chronic alcohol intake is associated with an increase in fasting plasma high density lipoproteins (HDL). To study alcohol's acute effects on plasma lipoproteins, we measured plasma lipoprotein concentrations and activities of postheparin plasma lipases in nine normolipemic males after ingestion of 40 g of ethanol (as whiskey). After alcohol there was no change in lipoprotein lipase activity but hepatic lipase was decreased to 67% of baseline at 6 hr. There were associated increases in HDL phospholipids (12 mg/dl) and cholesterol (10 mg/dl) resulting in prominence of larger, lipid-enriched HDL particles. Changes were most pronounced in the HDL3 and HDL2a subclasses. Very low density lipoprotein (VLDL) phospholipids and cholesterol were also increased by 13 and 9 mg/dl, respectively, with no significant change in triglycerides. Changes in lipoproteins and lipase were largely reversed 10 hr after alcohol intake. The transient increases in VLDL and HDL lipids after alcohol may result in part from acute inhibition of hepatic lipase activity. The results suggest a role of hepatic lipase in the catabolism of phospholipids of VLDL and possibly HDL.     

Defects of the LDL receptor in WHHL transgenic rabbits lead to a marked accumulation of plasma lipoprotein[a

In this study, we created LDL receptor (LDLr) defective (WHHL) transgenic rabbits expressing human apo[a] to examine whether LDLr mediates the Lp[a] clearance from the plasma. By crossbreeding WHHL rabbits with human apo[a] transgenic rabbits, we obtained two groups of human apo[a] transgenic rabbits with defective LDLr functions: apo[a](1/0) WHHL heterozygous (LDLr(+/-) and apo[a](+/0) WHHL homozygous (LDLr(-/-) rabbits. The lipid and lipoprotein levels of human apo[a] WHHL rabbits were compared to those of human apo[a] transgenic rabbits with normal LDLr functions (LDLr(+/+). The apo[a] production rate was evaluated by analyzing apo[a] mRNA expression in the liver, the major site for apo[a] synthesis in transgenic rabbits. We found that pre-beta lipoproteins were markedly increased accompanied by a 2-fold increase in the plasma Lp[a] in apo[a](+/0)/LDLr(+/-) rabbits and a 4.2-fold increase in apo[a](+/0)/LDLr(-/-) rabbits compared with that in apo[a](+/0) rabbits with normal LDLr function. In apo[a](+/0)/LDLr(-/-) rabbits, there was a marked increase in plasma total cholesterol and triglycerides, as was found in their counterpart non-transgenic WHHL rabbits. Northern blot analysis revealed that hepatic apo[a] expression in WHHL transgenic rabbits was similar to that in LDLr(+/+) transgenic rabbits, suggesting the accumulation of plasma Lp[a] in WHHL transgenic rabbits was not due to increased apo[a] synthesis.In conclusion, absence of a functional LDLr leads to a marked accumulation of plasma Lp[a] in human apo[a] transgenic WHHL rabbits and LDLr may participate in the catabolism of Lp[a] in rabbits.