Guinea pig very low density lipoproteins are a good substrate for lipoprotein lipase.

In contrast to plasma from most other animals, guinea pig plasma causes little or no stimulation of lipoprotein lipase activity. Very low density lipoproteins (VLDL) isolated by ultracentrifugation of guinea pig serum caused a definite stimulation of lipase activity, whereas the infranatant inhibited the activity. Gel filtration in 5 M guanidinium hydrochloride of delipidated VLDL demonstrated that the activation was caused by a low molecular weight protein. The VLDL themselves were hydrolized at similar rates as human VLDL both by guinea pig and by bovine lipoprotein lipases. Thus, guinea pig VLDL contain an activator for lipoprotein lipase analogous to that in other animals and there is enough of the activator to support rapid hydrolysis of the VLDL lipids by the lipase.     

Mechanisms by which lipoprotein lipase alters cellular metabolism of lipoprotein(a), low density lipoprotein, and nascent lipoproteins. Roles for low density lipoprotein receptors and heparan sulfate proteoglycans.

We sought to investigate effects of lipoprotein lipase (LpL) on cellular catabolism of lipoproteins rich in apolipoprotein B-100. LpL increased cellular degradation of lipoprotein(a) (Lp(a)) and low density lipoprotein (LDL) by 277% +/- 3.8% and 32.5% +/- 4.1%, respectively, and cell association by 509% +/- 8.7% and 83.9% +/- 4.0%. The enhanced degradation was entirely lysosomal. Enhanced degradation of Lp(a) had at least two components, one LDL receptor-dependent and unaffected by heparitinase digestion of the cells, and the other LDL receptor-independent and heparitinase-sensitive. The effect of LpL on LDL degradation was entirely LDL receptor-independent, heparitinase-sensitive, and essentially absent from mutant Chinese hamster ovary cells that lack cell surface heparan sulfate proteoglycans. Enhanced cell association of Lp(a) and LDL was largely LDL receptor-independent and heparitinase-sensitive. The ability of LpL to reduce net secretion of apolipoprotein B-100 by HepG2 cells by enhancing cellular reuptake of nascent lipoproteins was also LDL receptor-independent and heparitinase-sensitive. None of these effects on Lp(a), LDL, or nascent lipoproteins required LpL enzymatic activity. We conclude that LpL promotes binding of apolipoprotein B-100-rich lipoproteins to cell surface heparan sulfate proteoglycans. LpL also enhanced the otherwise weak binding of Lp(a) to LDL receptors. The heparan sulfate proteoglycan pathway represents a novel catabolic mechanism that may allow substantial cellular and interstitial accumulation of cholesteryl ester-rich lipoproteins, independent of feedback inhibition by cellular sterol content.     

Monoacylglycerol accumulation in low and high density lipoproteins during the hydrolysis of very low density lipoprotein triacylglycerol by lipoprotein lipase.

We have demonstrated that low and high density lipoproteins from monkey plasma are capable of accepting and accumulating monoacylglycerol that is formed by the action of lipoprotein lipase on monkey lymph very low density lipoproteins. Furthermore, the monoacylglycerol that accumulates in both low and high density lipoproteins is not susceptible to further hydrolysis by lipoprotein lipase but is readily degraded by the monoacylglycerol acyltransferase of monkey liver plasma membranes. These observations suggest a new mechanism for monoacylglycerol transfer from triacylglycerol rich lipoproteins to other lipoproteins. In addition, the finding that monoacylglycerol bound to low and high density lipoprotein is degraded by the liver enzyme but not lipoprotein lipase lends support to the hypothesis that there are distinct and consecutive extrahepatic and hepatic stages in the metabolism of triacylglycerol in plasma lipoproteins.     

Heparin binding to lipoprotein lipase and low density lipoproteins

Heparin was fractionated on an affinity column of bovine milk lipoprotein lipase (LpL) immobilized to Affi-Gel-15. The bound heparin, designated high-reactive heparin (HRH), enhanced LpL activity, presumably by stabilizing the enzyme against denaturation. The unbound heparin fraction had no observable effect on the initial rate of enzyme activity. However, at longer times of incubation there was inhibition of LpL activity. LpL-specific HRH also showed a high, Ca²⁺-dependent precipitating activity towards human plasma low density lipoproteins (LDL). Since LpL and LDL both bind to heparin-like molecules at the surface of the arterial wall, we suggest that their similar heparin-binding specificity may have physiological consequences as it relates to the development of atherosclerosis.

Heparin binding Lipoprotein lipase LDL Apolipoprotein Lipolysis     

Lipoprotein lipase-mediated lipolysis of very low density lipoproteins increases monocyte adhesion to aortic endothelial cells.

Lipoprotein lipase (LPL) bound to vascular endothelial cells hydrolyses triglycerides in plasma lipoproteins. To explore the role of LPL in atherogenesis, the effect of LPL-mediated lipolysis of very low density lipoproteins (VLDL) on monocyte adhesion to endothelial cells was examined. Adhesion of U937 monocytes to porcine aortic endothelial cells that were incubated with VLDL and purified bovine milk LPL was markedly higher than endothelial cells that were incubated with VLDL alone. The increase in monocyte adhesion obtained with VLDL was dependent on the concentration of the lipoprotein, monocyte dose and time of incubation. The increase in adhesion correlated with generation of free fatty acids from the hydrolysis of triglycerides in VLDL by LPL. Furthermore, direct addition of oleic acid to endothelial cells also increased adhesion of monocytes. We postulate that LPL-derived lipolytic products increase monocyte adhesion to vascular endothelium and thereby promote atherogenesis.     

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.     

Oxidized low density lipoproteins regulate synthesis of monkey aortic smooth muscle cell proteoglycans that have enhanced native low density lipoprotein binding properties

Oxidized low density lipoproteins (Ox-LDL) affect several biological processes involved in atherogenesis. However, it is not known whether Ox-LDL can regulate proteoglycan expression and thus affect arterial wall lipoprotein retention. This study evaluated whether Ox-LDL, as compared with native LDL, regulates proteoglycan expression by monkey arterial smooth muscle cells in vitro and whether proteoglycans synthesized in the presence of Ox-LDL exhibit altered lipoprotein binding properties. Ox-LDL stimulated glycosaminoglycan synthesis, as measured by (35)SO(4) incorporation, by 30-50% over that of native LDL. The effect was maximal after 72 h of exposure to 5 microg/ml of Ox-LDL. The molecular sizes of versican, biglycan, and decorin increased in response to Ox-LDL, as indicated by size exclusion chromatography and SDS-polyacrylamide gel electrophoresis. These effects could be mimicked by the lipid extract of Ox-LDL. These size increases were largely due to chain elongation and not to alterations in the ratio of (35)SO(4) to [(3)H]glucosamine incorporation. Affinity chromatography indicated that Ox-LDL stimulated the synthesis of proteoglycans with high affinity for native LDL. Ox-LDL also specifically stimulated mRNA expression for biglycan (but not versican or decorin), which was correlated with increased expression of secreted biglycan. Thus, Ox-LDL may influence lipoprotein retention by regulating synthesis of biglycan and also by altering glycosaminoglycan synthesis of vascular proteoglycans so as to enhance lipoprotein binding properties.     

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.     

Lipolysis of ApoC-II deficient very low density lipoproteins: enhancement of lipoprotein lipase action by synthetic fragments of apoC-II.

Enzymic hydrolysis of triacylglycerol has been studied with very low density lipoproteins from an individual with a genetically determined absence of apoC-II, the activator apoprotein for lipoprotein lipase. Normal rates of ester cleavage by purified bovine milk lipoprotein lipase can be achieved $\text{in}\text{vitro}$ with native apoC-II and by three shorter synthetic peptides, apoC-II(55–78), apoC-II(50–78) and apoC-II(43–78), which contain part of the carboxyl terminal third of the native apoprotein. At 0.5 μM concentration, all peptides produced a 7-fold activation. ApoC-II(43–78), but not apoC-II(50–78) or apoC-II(55–78), could bind VLDL as shown by separation of unbound ¹²⁵I peptides and the lipoproteins. Thus, residues 43–50 of apoC-II are part of a lipid binding region. High affinity binding of apoC-II peptides to the lipoprotein substrate is not obligatory for activation of lipoprotein lipase.     

Heparan sulfate proteoglycans are involved in opiate receptor-mediated cell migration

Opioid receptors are expressed in cells of the immune system, and potent immunomodulatory effects of their natural and synthetic ligands have been reported. In some studies, the opiate receptor antagonist naloxone itself displayed immunomodulatory actions. We investigated effects of naloxone on leukocyte chemotaxis. Cell migration was tested in micropore filter assays using modified Boyden chambers, and receptor expression was investigated using radiolabel binding assays. Naloxone induced peripheral blood nonadherent mononuclear cell and neutrophil chemotaxis at nanomolar concentrations and deactivated their migration toward beta-endorphin, angiotensin II, somatostatin, or interleukin-8 but not toward RANTES, vasoactive intestinal peptide, or substance P. Ligand binding studies showed no alteration in the binding of interleukin-8 to neutrophils by naloxone. Cleavage of heparan sulfate from proteoglycans on the cells' surface completely inhibited chemotactic and deactivating properties of naloxone but not other attractants. Chemotactic properties were abolished by pretreating cells with heparinase, chondroitinase, sodium chlorate, and anti-syndecan-4 antibodies, indicating the involvement of syndecan-4. The extent of migration toward naloxone was diminished by pretreatment with dimethylsphingosine, a specific sphingosine kinase inhibitor. As syndecan-4 signaling in leukocyte chemotaxis involves activation of sphingosine kinase, results indicate that naloxone interacts with syndecan-4 function in cell migration and suggest a role for heparan sulfate proteoglycans as coreceptors to members of the delta-opiate receptor family.     

Lipoprotein lipase-mediated interactions of small proteoglycans and low-density lipoproteins

According to numerous studies low-density lipoproteins (LDL) are supposed to interact with the glycosaminoglycan chain(s) of proteoglycans, e.g. with decorin and biglycan, which themselves are subject to receptor-mediated endocytosis. We tested, therefore, whether complexes of LDL and small proteoglycans can be endocytosed by either the LDL- or the small proteoglycan uptake mechanism. However, neither was the endocytosis of LDL significantly influenced by proteoglycans nor that of proteoglycans by LDL. This negative result could be explained by the observation that in vitro complex formation takes place only in buffers of low ionic strength. Under physiological conditions additional molecules may be necessary for complex stabilization. Lipoprotein lipase (LpL) which binds LDL was also able to interact with high affinity with decorin and its glycosaminoglycan-free core protein, both interactions being heparin-sensitive. Regardless of the presence or absence of LDL, LpL stimulated the endocytosis of decorin 1.5-fold, whereas LpL mediated a 4-fold stimulation of LDL uptake in the absence of decorin. No significant additional effect was seen in the presence of small concentrations of proteoglycans whereas in the presence of 1 microM decorin the endocytosis of [125I]LDL was reduced in normal as well as in LDL receptor-deficient fibroblasts. These observations could best be explained by assuming that LpL/LDL complexes are internalized upon binding to membrane-associated heparan sulphate and that small proteoglycans interfere with this process. It could not be ruled out, however, that a small proportion of the complexes is also taken up by the small proteoglycan receptor.     

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.     

Studies on pig serum lipoproteins. VI. Surface charge of very low density lipoprotein.

The surface electric charge of pig serum very low density lipoprotein (VLDL) is described. By isoelectric focusing VLDL was separated into at least 3 fractions having different isoelectric points and polypeptide distributions. The ultracentrifugal and electron microscopic results indicate that the VLDL was not drastically denatured by Ampholine.     

Structure of egg yolk very low density lipoprotein. Polydispersity of the very low density lipoprotein and the role of lipovitellenin in the structure

Egg yolk very low density lipoprotein contained on the average 75% of lipid which could be extracted by ether and 25% of a residual lipoprotein, the classical lipovitellenin. The ether-extracted lipid was composed of 75% triglycerides, 7% sterols, 2% mono- and diglycerides, and 16% phospholipids. Lipovitellenin contained 48% lipid composed of 87% phospholipids, 11% triglycerides, and 2% sterols. The protein vitellenin was composed for the most part of units of 74,000 and 270,000 daltons molecular weight.Egg yolk very low density lipoprotein is polydisperse. Preparative ultracentrifugation separated it into six fractions of different average molecular size, and gel chromatography separated it into five. The fractions of larger molecular size contained more lipid and triglyceride than did the fractions of smaller molecular size. The proteins of the fractions appeared to be similar.Egg yolk very low density lipoproteins appear to be a series of molecules composed of cores of lipid of varying sizes with each core surrounded by a layer of lipovitellenin, which is composed principally of glycoprotein and phospholipid.     

Heparan sulphate and the binding of lipoprotein lipase to porcine thoracic aorta endothelium

Purified bovine milk lipoprotein lipase was shown to bind to intact porcine aortic endothelium in a specific, saturable fashion. The binding was reversed by exogenous heparin. A single class of binding sites was involved and at saturation 1.24?10¹¹ molecules of lipoprotein lipase / cm² were bound. This represents 0.51?10⁶ enzyme molecules per endothelial cell at a density of 1.2?10³ molecules / μm². The enzyme binding was reduced by prior trypsinisation of the endothelial surface under conditions that removed cell surface glycosaminoglycan chains. The porcine endothelium was shown to have available at its surface 5.4?10¹¹ chains of heparan sulphate plus heparin-like glycosaminoglycans / cm². Such as excess suggests that lipoprotein lipase may interact with approximately one in four of the available heparan sulphate chains.     

Unesterified fatty acids inhibit the binding of low density lipoproteins to the human fibroblast low density lipoprotein receptor

Micromolar concentrations of oleate were found to inhibit reversibly the binding of low density lipoprotein (LDL) to the human fibroblast LDL receptor. The decrease in LDL binding caused a parallel reduction of both 125I-LDL uptake and degradation at 37 degrees C. At 4 degrees C, oleate was also found to displace 125I-LDL already bound to the LDL receptor. The effect of oleate was rapid, reaching 70-80% of maximum displacement with 5-10 min of incubation, and was closely correlated to oleate-albumin molar ratios. Partition analysis of unesterified fatty acids between cells and LDL showed that the inhibitory effect of oleate resulted mainly from an interaction of unesterified fatty acids with the cell surface rather than with the LDL particles. Using different unesterified fatty acids and fatty acid analogs, we found that the inhibitory effect was modulated by both the length and the conformation of the monomeric carbon chain and was directly dependent on the presence of a negative charge on the carboxylic group. At 4 degrees C, the inhibitory effect of oleate never exceeded half of maximum binding capacity. This limitation was associated with the ability of oleate to interact only with part of the population of LDL receptors which spontaneously recycles in the absence of ligand, as demonstrated by the fact that oleate did not induce any reduction of LDL binding after cell treatment with monensin in the absence of LDL. Our results indicate that unesterified fatty acids could participate in the control of LDL catabolism in vivo by direct modulation of the ability of LDL receptor to bind LDL.