Transfer of newly made triglyceride from hepatocytes to preexisting extracellular very low density lipoproteins

Previous in vivo studies suggested a new model to describe the metabolism of very low density lipoproteins (VLDL). It was hypothesized that some of the lipoprotein triglyceride was transferred directly from hepatocytes and intestinal mucosal cells into preexisting extracellular VLDL particles. These studies employ an in vitro system to test this hypothesis. Isolated rat liver cells containing newly made radioactive triglyceride were prepared. These cells were incubated in medium to which exogenous VLDL had or had not been added. The presence of extracellular VLDL (rat or human) stimulated the transfer of labeled triglyceride out of the liver cells. This triglyceride was recovered in the medium's VLDL (as determined by its density and its precipitability by MnCl2-heparin or by anti-apoprotein B). Although these studies focussed on VLDL, preliminary data showed that similar triglyceride transfer occurred in the presence of the other apoprotein B containing lipoprotein, low density lipoprotein (LDL). However, in the presence of equivalent amounts of LDL, this triglyceride transfer was less than that seen in the presence of exogenous VLDL. Furthermore, the increased triglyceride released in the presence of LDL occurred entirely in the d less than 1.006 fraction of the medium. That released in the presence of VLDL was recovered in the d greater than 1.006 fraction. Hence, we conclude that the transfer of the newly made triglyceride was from the cell to the extracellular lipoprotein that had been added to the medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hepatic triglyceride lipase promotes low density lipoprotein receptor-mediated catabolism of very low density lipoproteins in vitro.

We demonstrate here that hepatic triglyceride lipase (HTGL) enhances VLDL degradation in cultured cells by a LDL receptor-mediated mechanism. VLDL binding at 4 degrees C and degradation at 37 degrees C by normal fibroblasts was stimulated by HTGL in a dose-dependent manner. A maximum increase of up to 7-fold was seen at 10 microg/ml HTGL. Both VLDL binding and degradation were significantly increased (4-fold) when LDL receptors were up-regulated by treatment with lovastatin. HTGL also stimulated VLDL degradation by LDL receptor-deficient FH fibroblasts but the level of maximal degradation was 40-fold lower than in lovastatin-treated normal fibroblasts. A prominent role for LDL receptors was confirmed by demonstration of similar HTGL-promoted VLDL degradation by normal and LRP-deficient murine embryonic fibroblasts. HTGL enhanced binding and internalization of apoprotein-free triglyceride emulsions, however, this was LDL receptor-independent. HTGL-stimulated binding and internalization of apoprotein-free emulsions was totally abolished by heparinase indicating that it was mediated by HSPG. In a cell-free assay HTGL competitively inhibited the binding of VLDL to immobilized LDL receptors at 4 degrees C suggesting that it may directly bind to LDL receptors but may not bind VLDL particles at the same time.We conclude that the ability of HTGL to enhance VLDL degradation is due to its ability to concentrate lipoprotein particles on HSPG sites on the cell surface leading to LDL receptor-mediated endocytosis and degradation.     

In vivo transfer of cholesteryl esters from high density lipoproteins to very low density lipoproteins in man.

The fate of cholesteryl esters in high density lipoprotein (HDL) was studied to determine whether the transfer of esterified cholesterol from HDL to other plasma lipoproteins occurred to a significant extent in man. HDL cholesteryl ester, labelled in vitro with [3H] cholesterol, was injected into human subjects. Labelling of cholesteryl esters in very low density (VLDL) occurred rapidly and by 3 h, the esterified cholesterol in VLDL reached peak specific radioactivity. The removal rate of cholesteryl esters from HDL appeared to be exponential and of the order of 0.2/h; calculation of the apparent flux was about 150 mg/h which approximates reported values for total cholesterol esterification in human plasma in vivo. The rapid rate of labelling of VLDL from HDL suggests that the transfer of HDL cholesteryl esters to VLDL may represent a significant pathway for the disposal of HDL cholesterol.     

Lipoprotein lipase enhances the cholesteryl ester transfer protein-mediated transfer of cholesteryl esters from high density lipoproteins to very low density lipoproteins

These studies were undertaken to examine the effects of lipoprotein lipase (LPL) and cholesteryl ester transfer protein (CETP) on the transfer of cholesteryl esters from high density lipoproteins (HDL) to very low density lipoproteins (VLDL). Human or rat VLDL was incubated with human HDL in the presence of either partially purified CETP, bovine milk LPL or CETP plus LPL. CETP stimulated both isotopic and mass transfer of cholesteryl esters from HDL into VLDL. LPL caused only slight stimulation of cholesteryl ester transfer. However, when CETP and LPL were both present, the transfer of cholesteryl esters from HDL into VLDL remnants was enhanced 2- to 8-fold, compared to the effects of CETP alone. The synergistic effects of CETP and LPL on cholesteryl ester transfer were more pronounced at higher VLDL/HDL ratios and increased with increasing amounts of CETP. In time course studies the stimulation of cholesteryl ester transfer activity occurred during active triglyceride hydrolysis. When lipolysis was inhibited by incubating LPL with either 1 M NaCl or 2 mM diethylparanitrophenyl phosphate, the synergism of CETP and LPL was reduced or abolished, and LPL alone did not stimulate cholesteryl ester transfer. These experiments show that LPL enhances the CETP-mediated transfer of cholesteryl esters from HDL to VLDL. This property of LPL is related to lipolysis.     

The N-terminal 17% of apoB binds tightly and irreversibly to emulsions modeling nascent very low density lipoproteins

The N-terminal 17% of apolipoprotein B (apoB-17) readily associates with dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles (MLV) to form large (240-A diameter) discoidal particles. Because apoB is normally secreted with triacylglycerol (TAG)-rich lipoproteins, we studied the binding of apoB-17 to triolein-rich emulsions modeling nascent TAG-rich very low density-like lipoproteins. Emulsions with the following composition (by weight) were prepared: 85--89% triolein, 1.1--1.4% cholesterol, and 10--14% phosphatidylcholines (PC) including either egg yolk (EY)-, dimyristoyl (DM)-, or dipalmitoyl (DP)-PC representing (at 25 degrees C), respectively, a fluid surface, a surface at transition, and a mainly solid surface. The respective sizes were 1,260 +/- 500, 1,070 +/- 450, and 830 +/- 300 A mean diameter. The emulsions were incubated with conditioned medium containing apoB-17, and then reisolated by ultracentrifugation. Analysis of the emulsion-bound proteins by gel electrophoresis showed that all three emulsions bound primarily apoB-17. The DPPC emulsions bound more apoB-17 than EYPC or DMPC emulsions. Immunoaffinity-purified apoB-17 exhibited saturable, high affinity binding to EYPC and DPPC emulsions. The respective K(d) values were 32 +/- 23 and 85 +/- 27 nM and capacities (N) were 10 and 58 molecules of apoB-17 per particle. When apoB-17 bound to emulsions was incubated with DMPC MLV at 26 degrees C for 18 h, it remained bound to the emulsions, indicating that once bound to these emulsions it is unable to exchange off and solubilize DMPC into discs. In contrast, apoE-3 bound to emulsions dissociated from the emulsions when incubated with DMPC MLV and formed discs.Thus, apoB-17 binds strongly and irreversibly to emulsions modeling nascent lipoproteins. It therefore may play an important role in the stabilization of nascent VLDL and chylomicrons.- Herscovitz, H., A. Derksen, M. T. Walsh, C. J. McKnight, D. L. Gantz, M. Hadzopoulou-Cladaras, V. Zannis, C. Curry, and D. M. Small. The N-terminal 17% of apoB binds tightly and irreversibly to emulsions modeling nascent very low density lipoproteins. J. Lipid Res. 2001. 42: 51;-59.     

Interactions of high density lipoproteins with very low and low density lipoproteins during lipolysis

Interactions of high density lipoproteins (HDL) with very low (VLDL) and low (LDL) density lipoproteins were investigated during in vitro lipolysis in the presence of limited free fatty acid acceptor. Previous studies had shown that lipid products accumulating on lipoproteins under these conditions promote the formation of physical complexes between apolipoprotein B-containing particles (Biochim. Biophys. Acta, 1987. 919: 97-110). The presence of increasing concentrations of HDL or delipidated HDL progressively diminished VLDL-LDL complex formation. At the same time, association of HDL-derived apolipoprotein (apo) A-I with both VLDL and LDL could be demonstrated by autoradiography of gradient gel electrophoretic blots, immunoblotting, and apolipoprotein analyses of reisolated lipoproteins. The LDL increased in buoyancy and particle diameter, and became enriched in glycerides relative to cholesterol. Both HDL2 and HDL3 increased in particle diameter, buoyancy, and relative glyceride content, and small amounts of apoA-I appeared in newly formed particles of less than 75 A diameter. Association of apoA-I with VLDL or LDL could be reproduced by addition of lipid extracts of lipolyzed VLDL or purified free fatty acids in the absence of lipolysis, and was progressively inhibited by the presence of increasing amounts of albumin. We conclude that lipolysis products promote multiple interactions at the surface of triglyceride-rich lipoproteins undergoing lipolysis, including physical complex formation with other lipoprotein particles and transfers of lipids and apolipoproteins. These processes may facilitate remodeling of lipoproteins in the course of their intravascular metabolism.     

Plasma phospholipid transfer protein enhances transfer and exchange of phospholipids between very low density lipoproteins and high density lipoproteins during lipolysis

In order to determine the effects of a plasma phospholipid transfer protein on the transfer of phospholipids from very low density lipoproteins (VLDL) to high density lipoproteins (HDL) during lipolysis, biosynthetically labeled rat 32P-labeled VLDL was incubated with human HDL3 and bovine milk lipoprotein lipase (LPL) in the presence of the plasma d greater than 1.21 g/ml fraction or a partially purified human plasma phospholipid transfer protein (PTP). The addition of either the PTP or the d greater than 1.21 g/ml fraction resulted in a 2- to 3-fold stimulation of the transfer of phospholipid radioactivity from VLDL into HDL during lipolysis. In the absence of LPL, the PTP caused a less marked stimulation of transfer of phospholipid radioactivity. Both the d greater than 1.21 g/ml fraction and the PTP enhanced the transfer of VLDL phospholipid mass into HDL, but the percentage transfer of phospholipid radioactivity was greater than that of phospholipid mass, suggesting stimulation of both transfer and exchange processes. Stimulation of phospholipid exchange was confirmed in experiments where PTP was found to augment transfer of [14C]phosphatidylcholine radioactivity from HDL to VLDL during lipolysis. In experiments performed with human VLDL and human HDL3, both the d greater than 1.21 g/ml fraction and the PTP were found to stimulate phospholipid mass transfer from VLDL into HDL during lipolysis. Analysis of HDL by non-denaturing polyacrylamide gradient gel electrophoresis showed that enhanced lipid transfer was associated with only a slight increase in particle size, suggesting incorporation of lipid by formation of new HDL particles. In conclusion, the plasma d greater than 1.21 g/ml fraction and a plasma PTP enhance the net transfer of VLDL phospholipids into HDL and also exchange of the phospholipids of VLDL and HDL. Both the transfer and exchange activities of PTP are stimulated by lipolysis.     

Effect of human Apo AIV against lipid peroxidation of very low density lipoproteins

Recent studies have demonstrated that Apo AIV exerts a protective effect against atherosclerosis. Moreover, Qin et al. (Am. J. Physiol. 274 (1998) H1836) have demonstrated that Apo AIV, isolated from rat plasma, exerts an inhibitory effect against Cu(2+)-induced lipid peroxidation of intestinal lymph and LDL. The aim of the study was to investigate whether human Apo AIV exerts a protective effect against Cu(2+)-induced lipid peroxidation. Our results demonstrated that human Apo AIV exerted an inhibitory effect against Cu(2+) and AAPH induced lipid peroxidation of VLDL, as shown by the lower increase in the levels of TBARS and conjugated dienes in lipoproteins preincubated with Apo AIV. In addition, the tryptophan (Trp) and probe 2-(dimethylamino)-6-lauroylnaphthalene (Laurdan) fluorescence studies demonstrated that the modifications of spectral properties in both lipoproteins preincubated with Apo AIV were lower with respect to ox-lipoproteins, suggesting that Apo AIV prevents the modification of physico-chemical properties due to peroxidation.     

Influence of prostaglandins on the lipid transfer between human high density and low density lipoproteins

Prostaglandin (PG) E₁ was demonstrated to stimulate the transfer of phosphatidylcholine and cholesterol esters from human high density lipoproteins (HDL₃) to low density lipoproteins (LDL). The enhancement effect of PGE₁, on the interlipoprotein lipid transfer was seen at low PG concentrations under conditions of spontaneous exchange as well as in the presence of lipoprotein-depleted plasma, or partly purified plasma lipid exchange protein. PGE₂ and PGF₂ showed no significant influence on the interlipoprotein lipid transfer. Evidence is presented suggesting that the PGE₁-induced stimulation of interlipoprotein lipid exchange results in enhancement of LCAT-catalyzed cholesterol esterification in plasma. It is proposed that the effect of PGE₁ is due to the previously described PGE₁-induced reorganization of the HDL surface [(1984) FEBS Lett. 173, 291-293] and that PG-lipoprotein interaction may be a factor regulating cholesterol homeostasis.     

Time-related changes in the synthesis and secretion of very low density,low density and high density lipoproteins by cultured rat hepatocytes

Lipoproteins in the three major density classes were isolated from the medium of cultured rat hepatocytes incubated in the absence of serum for periods ranging from 1 to 48 h. De novo synthesis was suggested by the cyclo-heximide-sensitive incorporation of [³H]leucine into the apolipoproteins of the secreted lipoproteins.Hepatocyte d < 1.006 and d 1.006−1.063 g/ml lipoproteins were similar to plasma very low density lipoprotein (VLDL) and low density lipoprotein (LDL), respectively, in chemical composition, morphology and apolipoprotein distribution. The isolation of plasma-like d 1.006−1.063 g/ml particles is evidence for the hepatic origin of rat LDL; however, whether these particles are synthesized directly or result from catabolism of secreted VLDL has not been determined. Spherical d 1.063−1.21 g/ml particles containing predominantly apolipoprotein A-I were isolated from the media. In contrast to plasma high density lipoprotein (HDL) the hepatocyte particles contained significant concentrations of triacylglycerol and apolipoproteins of M_r > 100000 and lacked apolipoprotein A-IV.The pattern of lipoprotein secretion was related to the time of incubation. After incubation for 1, 3 and 6.5 h, VLDL comprised approx. 56% of the total lipoprotein mass, LDL 20% and HDL 24%. After 17 and 48 h the VLDL concentration was greatly reduced (approx. 20% of the total mass) while LDL and HDL concentrations were increased (33 and 47% of the total, respectively). Exogenous sodium oleate resulted in a concentration-dependent stimulation of VLDL synthesis at longer incubation periods. The triacylglycerol content of the secreted LDL fraction was also significantly increased following sodium oleate addition and there was an increased number of 425–650 Å particles present, which may represent catabolic products of VLDL. Hepatocyte mono-layers which can be maintained in serum-free media for extended periods should be useful for studying regulation of hepatic metabolism of the three major lipoprotein classes.     

Altered ultrastructural morphology of self-aggregated low density lipoproteins: coalescence of lipid domains forming droplets and vesicles

Lipid droplets and vesicles can presumably be formed directly from lipoproteins in the extracellular space in atherosclerosis, but an in vitro demonstration of the phenomenon in the absence of cellular pathways has been lacking. Low density lipoproteins (LDL) are known to undergo self-aggregation after brief vortexing in vitro. In the present study, LDL aggregates were examined by electron microscopy, using new mordant techniques for lipid visualization, and by chemical analysis. Aggregation of LDL by vortexing is regularly accompanied by the formation of comparatively large lipid droplets (up to 600 nm diameter) and vesicles. Aggregates containing droplets and vesicles were formed after as little as 5 sec of vortexing, and LDL protein and cholesteryl ester were almost completely (95%) incorporated into aggregates after 4 min vortexing. Substantial fractions of phospholipid and unesterified cholesterol from the original LDL remained in solution even after 4 min vortexing, forming large multilamellar vesicles that did not adhere to the aggregated material. Spontaneous aggregates retrieved from LDL solutions after prolonged storage were also examined by electron microscopy, revealing similar lipid droplets and vesicles. The ultrastructural appearance of LDL aggregated in vitro is remarkably similar to the appearance of extracellular lipid deposits in atherosclerosis, lending credence to the hypothesis of direct extracellular formation of these deposits from lipoproteins.     

Omega-3 fatty acids alter lipoprotein subfraction distributions and the in vitro conversion of very low density lipoproteins to low density lipoproteins

The purpose of this study was to determine the effects of a fish oil concentrate (FOC) on the in vitro conversion of very low density lipoproteins (VLDL) to intermediate (IDL) and low density lipoproteins (LDL). Six hypertriglyceridemic patients were randomly allocated to receive either placebo (olive oil) or FOC (1 g/14 kg body weight/day) for 4 weeks in a crossover study with a 4-week washout period. The FOC provided 3 g of eicosapentaenoic + docosahexaenoic acid per 70 kg of body weight, and it lowered plasma triglyceride and VLDL cholesterol levels by 35% and 42%, respectively. Decreases in the largest particles (VLDL(1)) were primarily responsible, with no effect noted in smaller VLDL particles (VLDL(2) and VLDL(3)). The FOC increased LDL cholesterol levels by 25% (P < 0.06) but did not affect LDL particle size. VLDL(1) and VLDL(3) were incubated in vitro with human postheparin lipases. Although triglycerides from both types of VLDL were hydrolyzed to the same extent with both treatments, particles isolated during the FOC phase were more readily converted into IDL and LDL than were control particles. These data suggest that the marine omega3 fatty acids may enhance the propensity of VLDL to be converted to LDL, partly explaining the decreased VLDL and increased LDL levels in FOC-treated patients.     

Age-related decrease of dehydroepiandrosterone concentrations in low density lipoproteins and its role in the susceptibility of low density lipoproteins to lipid peroxidation

The incidence of atherosclerosis and related diseases increases with age. The aging process may enhance lipoprotein modification, which leads to an increase in the susceptibility of low density lipoprotein (LDL) and high density lipoprotein (HDL) to oxidation. Dehydroepiandrosterone (DHEA), the most abundant steroid hormone in humans, has been shown to have antiatherogenic effects. This hormone also decreases dramatically with age. In the present study, we were interested in determining the presence of DHEA/DHEAS (dehydroepiandrosterone sulfate) and changes in their concentrations in HDL and LDL lipoproteins with age. Moreover, we studied the susceptibility of LDL to oxidation with age in the presence or absence of vitamin E or DHEA. We demonstrated that vitamin E is unable to restore the decreased resistance to oxidation of LDL from elderly subjects to that of LDL obtained from young subjects. Furthermore, our results provide evidence that DHEA is an integral part of LDL and HDL and disappears to almost nondetectable levels during aging. The DHEA incorporated into the LDL from elderly subjects increased LDL resistance to oxidation in a concentration-dependent manner. The increased resistance provided by DHEA was higher than that with vitamin E. DHEA seems to act either by protecting vitamin E from disappearance from LDL under oxidation or by scavenging directly the free radicals produced during the oxidative process. Our results suggests that DHEA exerts an antioxidative effect on LDL, which could have antiatherogenic consequences. Careful clinical trials of DHEA replacement should determine whether this ex vivo effect could be translated into any measurable antiatherogenic (cardioprotective) action.     

Characterization of the chicken oocyte receptor for low and very low density lipoproteins

The chicken oocyte receptor for low and very low density lipoproteins has been identified and characterized. Receptor activity present in octyl-beta-D-glucoside extracts of oocyte membranes was measured by a solid phase filtration assay, and the receptor was visualized by ligand blotting. The protein had an apparent Mr of 95,000 in sodium dodecyl sulfate-polyacrylamide gels under nonreducing conditions and exhibited high affinity for apolipoprotein B-containing lipoproteins, but not for high density lipoproteins or lipoproteins in which lysine residues had been reductively methylated. Binding of lipoproteins was sensitive to EDTA, suramin, and treatment with Pronase. In these aspects, the avian oocyte system was analogous to the mammalian low density lipoprotein receptor in somatic cells. Furthermore, a structural relationship between the mammalian and avian receptors was revealed by immunoblotting: polyclonal antibodies directed against the purified bovine low density lipoprotein receptor reacted selectively with the 95-kDa chicken receptor present in crude oocyte membrane extracts.     

Evidence for the presence of low density and very low density lipoproteins in human amniotic fluid

Previous analysis of amniotic fluid (AF) noted only the presence of high density lipoprotein (HDL). In this study AF lipoprotein profile was examined using gel filtration column chromatography and Ouchterlony gel diffusion. Unlike previous studies which showed only the presence of HDL, we found significant amounts of low density lipoprotein (LDL) and very low density lipoprotein (VLDL). AF-LDL and AF-VLDL were identified by reactions with anti-h-apolipoprotein AI and AII antiserum and anti-h-apolipoprotein B-antiserum, respectively. Furthermore, bulk of the cholesterol mass was carried in VLDL (53.6 +/- 7.7%) and LDL (32.5 +/- 4.3%) with minor amounts (13.9 +/- 1.3%) in HDL fraction. It is concluded that human AF contains all three lipoproteins with most of the cholesterol being carried in very low density lipoprotein fraction.