Metabolic heterogeneity in the formation of low density lipoprotein from very low density lipoprotein in the rat: evidence for the independent production of a low density lipoprotein subfraction.

The formation of low density lipoprotein (LDL) from very low density lipoprotein (VLDL) was studied after injecting 14C-radiomethylated or 125I-radioiodinated VLDL into rats. VLDL and LDL B apoprotein specific radioactivity time curves were obtained after tetramethylurea extraction of the lipoproteins. In all experiments, the specific activity of LDL B apoprotein did not intercept the VLDL curve at maximal heights, suggesting that not all LDL B apoprotein is derived from VLDL B apoprotein. Further subfractionation of LDL into the Sf 12-20, 5-12, and 0-5 ranges showed that most (65%) LDL B apoprotein was present in the Sf 0-5 fraction and that only a small proportion (6-15%) of this fraction was derived from VLDL. However, the curves obtained for the Sf 12-20 and 5-12 subfractions were consistent with a precursor-product relationship in which all of these fractions were derived entirely from VLDL catabolism. These results contrasted strikingly with similar data obtained for normal humans in which all LDL is derived from VLDL. In the rat, it appears that most of the B apoprotein in the Sf 0-5 range, which contains 65% of the total LDL B apoprotein, enters the plasma independently of VLDL secretion.     

Structural heterogeneity of apoB-containing serum lipoproteins visualized using cryo-electron microscopy.

Cryo-electron microscopy was used to analyze the structure of lipoprotein particles in density gradient subfractions of human very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), and low density lipoprotein (LDL). Lipoproteins from a normolipidemic subject with relatively large and buoyant LDL (pattern A) and from a subject with a predominance of small dense LDL (pattern B) were compared. Projections of VLDL in vitreous ice were heterogeneous in size, but all were circular with a relatively even distribution of contrast. Selected projections of LDL, on the other hand, were circular with a high density ring or rectangular with two high density bands. Both circular and rectangular LDL projections decreased in average size with increasing subfraction density, but were found in all of 10 density gradient subfractions, both in pattern A and in pattern B profiles. Preparations of total IDL contained particles with the structural features of VLDL as well as particles resembling LDL. IDL particles resembling LDL were observed in specific density gradient subfractions in the denser region of the VLDL;-IDL density range. Within the group of IDL particles resembling LDL considerable heterogeneity was observed, but no structural features specific for the pattern A or pattern B lipoprotein profile were recognized.The observed structural heterogeneity of the apolipoprotein B-containing serum lipoproteins may reflect differences in the composition of these particles that may also influence their metabolic and pathologic properties.     

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.     

Isopropanol precipitation method for the determination of apolipoprotein B specific activity and plasma concentrations during metabolic studies of very low density lipoprotein and low density lipoprotein apolipoprotein B

A method has been described for the measurement of apoB concentration and specific activity in very low density lipoprotein (VLDL) and low density lipoprotein (LDL) during metabolic studies. For measurement of specific activity, apoB was separated from other apolipoproteins and lipids by precipitation in, and subsequent washing with, isopropanol. For determination of apoB concentration, equal volumes of lipoprotein and isopropanol were mixed, and the protein content of the apoB precipitate was measured by the difference between total lipoprotein protein and the protein measured in the supernatant. Evidence that there was no apoB solubilization in isopropanol and that precipitated apoB was virtually free of soluble apolipoproteins was obtained by electrophoresis. Lipid contamination of the apoB precipitate was less than 1%. The methods were applicable to VLDL, intermediate density lipoprotein (IDL), and LDL from normolipemic patients with protein concentrations between 187 micrograms/ml and 1898 micrograms/ml. The data obtained using isopropanol were highly correlated with those using tetramethylurea, and recoveries of apoB were similar. Furthermore, the isopropanol method has been demonstrated to yield consistent data for apoB specific activities in a study of VLDL, IDL, and LDL metabolism.     

Studies on the metabolism of apolipoprotein B in hypertriglyceridemic subjects using simultaneous administration of tritiated leucine and radioiodinated very low density lipoprotein

To study the metabolic pathways of apolipoprotein B (apoB), a series of studies were carried out in which both radioiodinated very low density lipoproteins (VLDL) and tritiated leucine were simultaneously injected into three hypertriglyceridemic subjects. The appearance and disappearance of tritium activity in VLDL apoB, intermediate density lipoprotein (IDL) apoB, and low density lipoprotein (LDL) apoB were followed as was the disappearance of iodine activity from VLDL and the appearance and disappearance of iodine activity in IDL apoB and LDL apoB. It was found that a delipidation chain could describe the kinetics of both endogenously and exogenously labeled VLDL. A slow component of VLDL was necessary to fit the VLDL 131I-labeled apoB data and was consistent with the observed VLDL [3H]apoB kinetics. In addition, the estimated rate of conversion of VLDL apoB to LDL exceeded that which appeared to pass through the measured IDL pools, suggesting that a fraction of the IDL was not directly observed. It was also found that a higher percentage of VLDL 131I-labeled apoB was converted to LDL apoB than was VLDL [3H]apoB. To evaluate possible causes of this apparent anomaly, simultaneous examination of all kinetic data was performed. This exercise resulted in the resolution of removal pathways from multiple compartments in the VLDL delipidation chain and the conversion of slowly metabolized VLDL to IDL and LDL. The wide spectrum of this loss pathway indicates that previous estimates of VLDL apoB production rate using the radioiodinated methodology probably represent lower bounds for the true physiologic variable. It is important to note that these direct losses were apparent only when the combination of endogenous and exogenous labeling was used.     

Metabolism of apoprotein B in selectively bred baboons with low and high levels of low density lipoproteins

Very low density lipoprotein (VLDL) and low density lipoprotein (LDL) apoprotein (apo)-B turnover rates were measured simultaneously by injecting 131I-labeled VLDL and 125I-labeled LDL into fasting baboons (Papio sp.) selectively bred for high serum cholesterol levels and having either low or high LDL levels. The radioactivities in VLDL, intermediate density lipoprotein (IDL), LDL apoB, and urine were measured at intervals between 5 min and 6 days. Kinetic parameters for apoB were calculated in each baboon fed a chow diet or a high cholesterol, high fat diet (HCHF). VLDL apoB residence times were similar in the two groups of animals fed chow; they were increased by HCHF feeding in high LDL animals, but not in low LDL animals. Production rates of VLDL apoB were decreased by the HCHF diet in both high and low LDL animals. Most of the radioactivity from VLDL apoB was transferred to IDL. However, a greater proportion of radioactivity was removed directly from IDL apoB in low LDL animals than in high LDL animals, and only about one-third appeared in LDL. In high LDL animals, a greater proportion of this radioactivity was converted to LDL (61.4 +/- 7.2% in chow-fed animals and 49.2 +/- 10.9% in animals fed the HCHF diet; mean +/- SEM, n = 5). Production rates for LDL apoB were higher in high LDL animals than those in low LDL animals on both diets. The HCHF diet increased residence times of LDL apoB without changing production rates in both groups. VLDL apoB production was not sufficient to account for LDL apoB production in high LDL animals, a finding that suggested that a large amount of LDL apoB was derived from a source other than VLDL apoB in these animals.     

Metabolic pathways of apolipoprotein B in heterozygous familial hypercholesterolemia: studies with a [3H]leucine tracer.

The kinetics of apolipoprotein B (apoB) were measured in seven studies in heterozygous, familial hypercholesterolemic subjects (FH) and in five studies in normal subjects, using in vivo tracer kinetic methodology with a [3H]leucine tracer. Very low density (VLDL) and low density lipoproteins (LDL) were isolated ultracentrifugally and LDL was fractionated into high and low molecular weight subspecies. ApoB was isolated, its specific radioactivity was measured, and the kinetic data were analyzed by compartmental modeling using the SAAM computer program. The pathways of apoB metabolism differ in FH and normal subjects in two major respects. Normals secrete greater than 90% of apoB as VLDL, while one-third of apoB is secreted as intermediate density lipoprotein IDL/LDL in FH. Normals lose 40-50% of apoB from plasma as VLDL/IDL, while FH subjects lose none, metabolizing all of apoB to LDL. In FH, there is also the known prolongation of LDL residence time. The leucine tracer, biosynthetically incorporated into plasma apoB, permits distinguishing the separate pathways by which the metabolism of apoB is channeled. ApoB synthesis and secretion require 1.3 h. ApoB is secreted by three routes: 1) as large VLDL where it is metabolized by a delipidation chain; 2) as a rapidly metabolized VLDL fraction converted to LDL; and 3) as IDL or LDL. ApoB is metabolized along two pathways. The delipidation chain processes large VLDL to small VLDL, IDL, and LDL. The IDL pathway channels nascent, rapidly metabolized VLDL and IDL particles into LDL. It thus provides a fast pathway for the entrance of apoB tracer into LDL, while the delipidation pathway is a slower route for channeling apoB through VLDL into LDL. LDL apoB is derived in almost equal amounts from both pathways, which feed predominantly into large LDL. Small LDL is a product of large LDL, and the major loss of LDL-apoB is from small LDL. Two features of apoB metabolism in FH, the major secretory pathway through IDL and the absence of a catabolic loss of apoB from VLDL/IDL, greatly facilitate measuring the metabolic channeling of apoB into LDL.     

Effects of bezafibrate on apolipoprotein B metabolism in type III hyperlipoproteinemic subjects

This study was designed to investigate the response of Type III hyperlipoproteinemic subjects to bezafibrate therapy. The metabolism of apolipoprotein B was examined in four lipoprotein subclasses of Sf 60-400 (large very low density lipoprotein (VLDL)), Sf 20-60 (small VLDL), Sf 12-20 (intermediate density lipoprotein (IDL)), and Sf 0-12 (low density lipoprotein (LDL)) before and during bezafibrate therapy. Treatment reduced the plasma concentration of VLDL and raised high density lipoprotein (HDL) cholesterol. There was no net change in LDL cholesterol or its associated apolipoprotein B. The decrease in plasma VLDL derived mainly from an inhibition of synthesis of both large and small subfractions which reduced the number of particles in the circulation without normalizing their lipid composition. Catabolism of the larger VLDL also increased, presumably as a result of lipoprotein lipase activation. Although the plasma concentration of LDL was unchanged, both its synthesis and catabolism were perturbed. Its fractional catabolic rate fell by 50%, but the impact that this would have had on its steady state level in the circulation was apparently blunted by a decrease in its synthesis from Sf 12-20 IDL. In the control phase of the study, most IDL apolipoprotein B was converted to LDL. Bezafibrate therapy channelled this material towards direct catabolism.     

Lipoprotein lipase-mediated sequestration of long-chain polyunsaturated triacylglycerols in serum LDL from normal and hypothyroid rats

Rat serum VLDL, unlike human, contains significant proportions of triacylglycerols with polyunsaturated C20 and C22 fatty acids. Hypothyroidism in this species is characterized by low levels of serum VLDL, the accumulation of LDL, elevated levels of lipoprotein lipase and depressed hepatic lipase activity. The hypothyroid rat thus represents an interesting model in which to study hepatic VLDL metabolism and the substrate specificity of lipoprotein lipase. This report shows that serum IDL and LDL in both euthyroid and hypothyroid rats contain progressively enhanced proportions of triacylglycerols with polyunsaturated C20 and C22 fatty acids when compared to VLDL. Hypothyroidism resulted in a decrease in the proportion of 22:6 fatty acid within the serum VLDL triacylglycerols when compared to euthyroid VLDL. Lipolysis of VLDL from euthyroid rats in vitro using the perfused rat heart system resulted in increases or sequestration of triacylglycerols containing long-chain polyunsaturated fatty acids within the IDL fraction similar to those seen in vivo. It is concluded that lipoprotein lipase-mediated hydrolysis of VLDL triacylglycerols and the conversion of VLDL to IDL and LDL in the rat results in a progressive sequestration of the longer-chain polyunsaturated triacylglycerol molecular species with the IDL and LDL.     

A rapid electrophoretic technique for identification of subunit species of apoproteins in serum lipoproteins

The denaturing solvent tetramethylurea (TMU) delipidates and quantitatively liberates the apoproteins of human serum high-density lipoprotein (HDL) in soluble form while virtually the whole apoprotein of human lowdensity lipoprotein (LDL) is precipitated. A fraction of the apoprotein of very low density lipoprotein (VLDL) which appears to represent its content of LDL-like protein (apo B) is precipitated by this reagent, while the remaining apoprotein species are liberated in soluble form.The dissociation of the soluble apoproteins from lipid by TMU obviates the need for time-consuming delipidation by organic solvents, permitting immediate electrophoretic analysis in polyacrylamide gels. Bands are observed with mobilities corresponding to those of all the major soluble polypeptide species isolated from serum lipoproteins by ion-exchange chromatography. The apparent distribution of these elements in the different classes of lipoproteins is in agreement with findings of studies employing chromatographic methods. The predominant apoprotein of HDL, which has been identified immunochemically in VLDL, appears to comprise less than 1% of the apoprotein of VLDL from normal serum.     

Effects of 1,2-cyclohexanedione modification on the metabolism of very low density lipoprotein apolipoprotein B: potential role of receptors in intermediate density lipoprotein catabolism

The conversion of very low density (VLDL) to low density lipoproteins (LDL) is a two-step process. The first step is mediated by lipoprotein lipase, but the mechanism responsible for the second is obscure. In this study we examined the possible involvement of receptors at this stage. Apolipoprotein B (apoB)-containing lipoproteins were separated into three fractions, VLDL (Sf 100-400), an intermediate fraction IDL (Sf 12-100), and LDL (Sf 0-12). Autologous 125I-labeled VLDL and 131I-labeled 1,2-cyclohexanedione-modified VLDL were injected into the plasma of four normal subjects and the rate of transfer of apoB radioactivity was followed through IDL to LDL. Modification did not affect VLDL to IDL conversion. Thereafter, however, the catabolism of modified apoB in IDL was retarded and its appearance in LDL was delayed. Hence, functional arginine residues (and by implication, receptors) are required in this process. Confirmation of this was obtained by injecting 125I-labeled IDL and 131I-labeled cyclohexanedione-treated IDL into two additional subjects. Again, IDL metabolism was delayed by approximately 50% as a result of the modification. These data are consistent with the view that receptors are involved in the metabolism of intermediate density lipoprotein.     

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

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

Direct determination of human and rabbit apolipoprotein B selectively precipitated with butanol-isopropyl ether

A method is described for the rapid, selective, and quantitative precipitation of apolipoprotein B from isolated hypercholesterolemic rabbit and human very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), and low density lipoproteins (LDL). Lipoprotein samples are heat-treated at 100 degrees C in 1% SDS. The denatured apoprotein solutions are then mixed briefly with two volumes of butanol-isopropyl ether 45:55 (v/v) to precipitate the apoB. The supernatant solutions, containing the non-apoB proteins and lipids, are removed and the apoB pellet is washed once with water. To determine apoB specific activity, the apoB pellet is resolubilized in 0.5 M NaOH by heating for 30 min at 120 degrees C. The hydrolyzed apoB protein is quantitated by fluorescence of a fluorescamine derivative. The precipitation of apoB is quantitative and selective: 99.5% of rabbit 125I-labeled LDL-apoB and 97.5% of human 125I-labeled LDL-apoB is precipitated and less than 5% of 125I-labeled HDL added to unlabeled VLDL, IDL, or LDL is precipitated. Triglyceride and cholesteryl ester contamination of the apoB pellet is less than 2% of their original radioactivities.     

Increased hepatic lipase activity and increased direct removal of very-low-density lipoprotein remnants in Watanabe heritable hyperlipidaemic (WHHL) rabbits treated with ethinyl oestradiol.

We studied the effects of ethinyl oestradiol on the serum concentrations and metabolism of very-low- and low-density lipoproteins (VLDL and LDL) in Watanabe heritable hyperlipidaemic (WHHL) homozygous rabbits, an animal model for familial hypercholesterolaemia. The results were compared with those in untreated homozygotes as well as in heterozygotes treated or not with ethinyl oestradiol. The gain in body weight was similar in all groups. Treatment with ethinyl oestradiol resulted in the homozygotes in an approx. 80% decrease in the concentrations of lipids and apoprotein B in the d less than 1.019 lipoprotein fraction; those in the LDL fraction did not change. In the heterozygotes, basal serum lipids and apoprotein B levels in the d less than 1.019 fraction were low; ethinyl oestradiol treatment especially affected the LDL fraction (cholesterol -84%, apoprotein B -64%). Turnover experiments with 125I-labelled VLDL revealed that, on treatment with ethinyl oestradiol, the fractional catabolic rate in homozygous rabbits increased 2-fold. The secretion rates of lipids and protein in the d less than 1.019 fraction as estimated after injection of Triton WR-1339 was not decreased. In homozygotes and heterozygotes increases in post-heparin hepatic lipase activity of 62 and 80% respectively were observed, with no changes in lipoprotein lipase activity. We conclude that ethinyl oestradiol induces in homozygous WHHL rabbits a direct removal of VLDL and VLDL remnants from the plasma, apparently due to an increase in hepatic lipase activity.     

Metabolism of apoB-100 in lipoproteins separated by density gradient ultracentrifugation in normal and Watanabe heritable hyperlipidemic rabbits

We have examined the capability of a previously developed compartmental model to explain the kinetics of radioiodinated apolipoprotein (apo) B-100 in very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), and low density lipoproteins (LDL) separated by density gradient ultracentrifugation after intravenous injection of radioiodinated VLDL into New Zealand white (NZW) and Watanabe heritable hyperlipidemic (WHHL) rabbits. Our model was developed primarily from kinetics in whole blood plasma of apoB-100 in particles with and without apoE after intravenous injection of large VLDL, total VLDL, IDL, and LDL. When the initial conditions for this model were assumed to be an intravenous injection of radiolabeled VLDL, the plasma VLDL and LDL simulations for NZW rabbits and the VLDL, IDL, and LDL simulations for WHHL rabbits were found to be inconsistent with the observed density gradient data. By adding a new pathway in the VLDL portion of the model for NZW rabbits and a new compartment in VLDL for WHHL rabbits, and by assuming some cross-contamination in the density gradient ultracentrifugal separations, it was possible to bring our model, which was based upon measurements of 125I-labeled apoB-100 in whole plasma, into conformity with the data obtained by density gradient ultracentrifugation. The relatively modest changes required in the model to fit the gradient ultracentrifugation data support the suitability of our approach to the kinetic analysis of the metabolism of apoB-100 in VLDL and its conversion to IDL and LDL based upon measurements of 125I-labeled apoB-100 in whole plasma after injection of radiolabeled VLDL, IDL, and LDL. Furthermore, the differences in kinetics observed by us between data from whole plasma and data from plasma submitted to ultracentrifugal separation from the same or similar animals highlight the fact that small variations that can occur in the separation of lipoprotein classes by buoyant density can lead to confusing results.     

Measurement of apolipoprotein B concentration in plasma lipoproteins by combining selective precipitation and mass spectrometry

The measurement of apolipoprotein B (apoB) in purified lipoproteins by immunological assays is subject to criticism because of denatured epitopes or immunoreactivity differences between purified lipoproteins and standard. Chemical methods have therefore been developed, such as the selective precipitation of apoB followed by quantification of the precipitate. In this study, we present the measurement of apoB concentration in lipoproteins purified by ultracentrifugation by combining isopropanol precipitation and gas chromatography/mass spectrometry. Very low density lipoprotein (VLDL; d < 1.006 g/mL); VLDL plus intermediate density lipoprotein (VLDL + IDL; d < 1.019 g/mL); and VLDL, IDL, and low density lipoprotein (VLDL + IDL + LDL; d < 1.063 g/mL) were purified by ultracentrifugation. Apolipoprotein B-100 was selectively precipitated by isopropanol. The leucine content of the pellet was then determined by gas chromatography/mass spectrometry, using norleucine as internal standard. Knowledge of the number of leucine molecules in one apoB-100 molecule makes it possible to calculate the plasma concentration of apoB in the various lipoprotein fractions. ApoB in IDL (d 1.006-1.019 g/mL) and LDL (d 1.019-1.063 g/mL) were then determined by subtracting VLDL-apoB from apoB in lipoproteins d < 1.019 and apoB in lipoproteins d < 1.019 g/mL from apoB in lipoproteins d < 1.063 g/mL, respectively. The isopropanol precipitate was verified as pure apoB (>97%) in lipoprotein fractions isolated from normo- and hyperlipidemic plasma and the method appeared reproducible.The combination of isopropanol precipitation and the GC/MS method appears therefore to be a precise and reliable method for kinetic and epidemiological studies.     

Lipoprotein secretion in lean and obese Zucker female rats in vivo and in a single-pass-perfused liver preparation

The plasma lipoprotein composition as well as lipoprotein synthesis and secretion were studied in vivo and in a single-pass-perfused liver preparation in lean and obese Zucker rats. Compared with their lean littermates the levels in the plasma of very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL) + low density lipoprotein (LDL) and high density lipoprotein (HDL) were increased 4-, 2- and 2.5 fold, respectively, in obese rats. In these rats both VLDL and IDL + LDL were enriched in triglycerides, while the HDL were enriched in cholesterol. Although the VLDL and IDL + LDL protein concentrations were the same in lean and obese rats, the HDL protein concentration was 3-fold greater in the obese rats. Both the lean and obese rats incorporated similar amounts of [14C]leucine into total liver protein. However, obese rats incorporated 2.5-fold and 6-fold more [14C]leucine into VLDL and HDL in vivo, 2.7-fold and 1.7 fold more [35S]methionine in VLDL and HDL present in the perfusate, than did lean rats. The perfusate [35S]S-labelled apoproteins (apo-B100, B48; apo-E, apo-AI, apo-AIV and apo-C) were separated by gel electrophoresis and identified by autoradiography. Incorporation of [3H]glycerol into liver, VLDL, IDL + LDL and HDL triglycerides was 2-, 48-, 13- and 1.5-fold higher in obese than in lean rats, respectively. The [3H]-labelled triglycerides in VLDL and IDL + LDL present in the perfusate was 5.4-fold and 4.4-fold more in obese rat. There was no difference in the incorporation of [3H]glycerol into triglycerides of perfusate HDL between the two genotypes of rats. Thus, the hypertriglyceridaemia observed in obese Zucker rats results from very high synthetic rates of both the lipid and protein moieties of plasma lipoproteins. Before this study, no report of the simultaneous triglycerides and protein synthesis in vivo and in a single-pass-perfused liver preparations had been reported.     

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

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

Metabolomic analysis of polar metabolites in lipoprotein fractions identifies lipoprotein-specific metabolic profiles and their association with insulin resistance

While the molecular lipid composition of lipoproteins has been investigated in detail, little is known about associations of small polar metabolites with specific lipoproteins. The aim of the present study was to investigate the profiles of polar metabolites in different lipoprotein fractions, i.e., very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL) and two sub-fractions of the high-density lipoprotein (HDL). The VLDL, IDL, LDL, HDL(2), and HDL(3) fractions were isolated from serum of sixteen individuals having a broad range of insulin sensitivity and characterized using comprehensive two-dimensional gas chromatography combined with time-of-flight mass spectrometry (GC×GC-TOFMS). The lipoprotein fractions had clearly different metabolite profiles, which correlated with the particle size and surface charge. Lipoprotein-specific associations of individual metabolites with insulin resistance were identified, particularly in VLDL and IDL fractions, even in the absence of such associations in serum. The results indicate that the polar molecules are strongly attached to the surface of the lipoproteins. Furthermore, strong lipoprotein-specific associations of metabolites with insulin resistance, as compared to their serum profiles, indicate that lipoproteins may be a rich source of tissue-specific metabolic biomarkers.     

A new micromethod for routine measurement of serum LDL-apolipoprotein B

A new method for low density lipoprotein (LDL) (d 1.019-1.063 g/ml)-apolipoprotein B (apoB) determination has been developed, based on the fact that very low density and intermediate density lipoproteins (VLDL and IDL) contain apolipoprotein C-I (apoC-I), whereas this apolipoprotein is apparently absent in LDL. VLDL and IDL were quantitatively precipitated with a monospecific anti-apoC-I antibody whereafter LDL-apoB in the supernatant was quantitated by Laurell rocket electrophoresis. Over a wide range of cholesterol and triglyceride values there was a linear correlation with LDL-apoB values measured after ultracentrifugation. The method would be useful for routine measurements, especially in children, since only 25 microliter of serum is required, and for making the diagnosis of hyperapobetalipoproteinemia, which at present is complicated.