Release of endothelial cell lipoprotein lipase by plasma lipoproteins and free fatty acids

Lipoprotein lipase (LPL) bound to the lumenal surface of vascular endothelial cells is responsible for the hydrolysis of triglycerides in plasma lipoproteins. Studies were performed to investigate whether human plasma lipoproteins and/or free fatty acids would release LPL which was bound to endothelial cells. Purified bovine milk LPL was incubated with cultured porcine aortic endothelial cells resulting in the association of enzyme activity with the cells. When the cells were then incubated with media containing chylomicrons or very low density lipoproteins (VLDL), a concentration-dependent decrease in the cell-associated LPL enzymatic activity was observed. In contrast, incubation with media containing low density lipoproteins or high density lipoproteins produced a much smaller decrease in the cell-associated enzymatic activity. The addition of increasing molar ratios of oleic acid:bovine serum albumin to the media also reduced enzyme activity associated with the endothelial cells. To determine whether the decrease in LPL activity was due to release of the enzyme from the cells or inactivation of the enzyme, studies were performed utilizing radioiodinated bovine LPL. Radiolabeled LPL protein was released from endothelial cells by chylomicrons, VLDL, and by free fatty acids (i.e. oleic acid bound to bovine serum albumin). The release of radiolabeled LPL by VLDL correlated with the generation of free fatty acids from the hydrolysis of VLDL triglyceride by LPL bound to the cells. Inhibition of LPL enzymatic activity by use of a specific monoclonal antibody, reduced the extent of release of 125I-LPL from the endothelial cells by the added VLDL. These results demonstrated that LPL enzymatic activity and protein were removed from endothelial cells by triglyceride-rich lipoproteins (chylomicrons and VLDL) and oleic acid. We postulate that similar mechanisms may be important in the regulation of LPL activity at the vascular endothelium.     

Identification and characterization of the endothelial cell surface lipoprotein lipase receptor.

The hydrolysis of triglycerides in plasma lipoproteins is mediated by lipoprotein lipase (LPL) that is bound to vascular endothelial cells. The specific endothelial cell surface protein(s) with which LPL associates has not been characterized. To identify this LPL binding protein(s), radioiodinated cell surface proteins from cultured bovine aortic endothelial cells were chromatographed using bovine LPL-Sepharose. A single radioiodinated protein of apparent molecular mass 220 kDa was specifically retained by the gel and eluted with 0.4 M NaCl. A LPL-binding protein of similar size was obtained after metabolic labeling of the cellular proteoglycans with 35SO4, indicating that the 220-kDa protein is a proteoglycan. After heparitinase or nitrous acid treatments the molecular mass of the LPL-binding protein decreased to approximately 50 kDa, suggesting that it contains heparin sulfate chains. A 220-kDa protein from the basal cell surface was also identified using LPL-Sepharose chromatography. 125I-LPL was cross-linked to the endothelial cell surface using ethylene glycobis (succinimidylsuccinate). A single ligand-receptor complex, approximately 350 kDa, was obtained. Heparin and unlabeled LPL decreased the cross-linking of radioiodinated LPL to the cell surface receptor. To examine whether the receptor mediates the internalization of cross-linked 125I-LPL, cells containing 125I-LPL complexed to the surface were incubated at either 37 or at 4 degrees C. The amount of 125I-LPL internalized by the cells was 74% greater at 37 degrees C than at 4 degrees C. This suggested that LPL cross-linked to the receptor was internalized in a temperature-dependent manner. Thus, a 220-kDa heparan sulfate proteoglycan functions as an endothelial cell surface receptor for LPL.     

Metabolism of endothelial cell-bound lipoprotein lipase. Evidence for heparan sulfate proteoglycan-mediated internalization and recycling

Lipoprotein lipase (LPL) hydrolyzes triglyceride in plasma lipoprotein primarily while bound to vascular endothelial cells. LPL metabolism by cultured endothelial cells was studied. Purified radioiodinated bovine LPL bound to porcine aortic endothelial cells at 4 degrees C with an association constant of 0.18 x 10(7) m-1. Analysis of the time course of LPL dissociation from endothelial cells at 4 degrees C yielded a dissociation rate constant of 3.9 x 10(-6)s-1. After 1 h at 37 degrees C, 28% of the LPL initially bound to the cell surface was no longer releasable by heparin or trypsin treatments, suggesting that LPL was internalized by the cells. Addition of heparin to the medium or pretreatment of the cells with heparinase markedly reduced the amount of LPL internalized, establishing a requirement for cell surface heparan sulfate proteoglycans in the process. When cells containing internalized LPL were incubated at 37 degrees C, a time-dependent increase in the amount of LPL in the medium and a corresponding decrease in LPL associated with the cells was found. This suggested that internalized LPL was released back into the medium. The catalytic activity, molecular size, and heparin-binding characteristics of the released LPL was similar to native LPL. Addition of either heparin, heparinase, or excess unlabeled LPL to prevent the rebinding of released 125I-LPL to the cell surface increased the amount of 125I-LPL present in the medium, suggesting that there is a process of recycling of 125I-LPL bound to the cell surface. Studies examining the effect of pH on dissociation of LPL from its binding site showed less dissociation of cell surface bound LPL at pH 5.5 compared with pH 7.4 and 8.5. These results suggest that even at acidic pH as in endocytotic vesicles, LPL remains bound to proteoglycans and this may facilitate the recycling of internalized LPL molecules.     

Transcytosis of lipoprotein lipase across cultured endothelial cells requires both heparan sulfate proteoglycans and the very low density lipoprotein receptor

Lipoprotein lipase (LPL), the major enzyme responsible for the hydrolysis of circulating lipoprotein triglyceride molecules, is synthesized in myocytes and adipocytes but functions while bound to heparan sulfate proteoglycans (HSPGs) on the luminal surface of vascular endothelial cells. This requires transfer of LPL from the abluminal side to the luminal side of endothelial cells. Studies were performed to investigate the mechanisms of LPL transcytosis using cultured monolayers of bovine aortic endothelial cells. We tested whether HSPGs and members of the low density lipoprotein (LDL) receptor superfamily were involved in transfer of LPL from the basolateral to the apical side of cultured endothelial cells. Heparinase/heparinitase treatment of the basolateral cell surface or addition of heparin to the basolateral medium decreased the movement of LPL. This suggested a requirement for HSPGs. To assess the role of receptors, we used either receptor-associated protein, the 39-kDa inhibitor of ligand binding to the LDL receptor-related protein and the very low density lipoprotein (VLDL) receptor, or specific receptor antibodies. Receptor-associated protein reduced (125)I-LPL and LPL activity transfer across the monolayers. When the basolateral surface of the cells was treated with antibodies, only anti-VLDL receptor antibodies inhibited transcytosis. Moreover, overexpression of the VLDL receptor using adenoviral-mediated gene transfer increased LPL transcytosis. Thus, movement of active LPL across endothelial cells involves both HSPGs and VLDL receptor.     

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.     

Identification of a heparin-releasable lipoprotein lipase binding protein from endothelial cells.

Triglycerides in circulating plasma lipoproteins are hydrolyzed by lipoprotein lipase (LPL) which is thought to bind to proteoglycans on the luminal endothelial cell surface. Previous studies from this laboratory using LPL-Sepharose affinity chromatography identified a 220-kDa LPL binding proteoglycan. Using ligand blotting with 125I-LPL, we now report a 116-kDa LPL binding protein in plasma membrane preparations of endothelial cells. 125I-LPL binding to this protein was abolished by addition of unlabeled LPL. When the cell surface of endothelial cells was labeled with biotin, a 116-kDa protein was biotinylated. Furthermore, the biotinylated 116-kDa protein bound to LPL-Sepharose and eluted with 0.4 M NaCl suggesting that the 116-kDa LPL binding protein is present on the cell surface. When detergent extracts of endothelial cells were applied to LPL-Sepharose in the presence of 0.15 M NaCl, the 116-kDa, but not the 220-kDa, protein still bound to LPL-Sepharose. The 116-kDa protein was not labeled with 35SO4 and eluted from DEAE-cellulose prior to proteoglycans, suggesting that it is not a proteoglycan. However, a 116-kDa endothelial cell surface protein was metabolically labeled with [35S]methionine. This protein was dissociated from the cell surface by incubating cells with heparin (50 units/ml)-containing buffer. After heparin treatment of endothelial cells, LPL binding to and internalization by the cells decreased greater than 70% compared to control cells. These results suggest that endothelial cells synthesize a heparin-releasable, high affinity 116-kDa LPL binding protein. We postulate that this protein is associated with proteoglycans on luminal endothelial surfaces and mediates LPL binding, internalization, and recycling. We name this protein hrp (heparin-releasable protein)-116.     

Differentiation-dependent expression of retinoid-binding proteins in BFC-1 beta adipocytes.

Recently, we demonstrated that adipose tissue plays an important role in retinol storage and retinol-binding protein (RBP) synthesis. Our data suggested that RBP expression in adipose tissue is dependent on the state of adipocyte differentiation. To examine this possibility, we explored the differentiation-dependent expression of RBP using BFC-1 beta preadipocytes, which can be stimulated to undergo adipose differentiation. Total RNA was isolated from undifferentiated (preadipocytes) and differentiated (adipocytes) BFC-1 beta cells and analyzed by Northern blotting. RBP mRNA was not detected in the preadipocytes, but considerable RBP mRNA was present in differentiated BFC-1 beta cells. In BFC-1 beta cells, induced to differentiate with insulin and thyroid hormone, RBP mRNA was first detected after 4 days, reached a maximum level by day 10, and remained at this maximum level for at least 2 more days. Cellular retinol-binding protein was expressed at low levels in the BFC-1 beta preadipocytes and the level of expression increased for 6 days after induction to differentiate and slowly declined on later days. Neither the maximum level of RBP expression nor the day on which this level was reached was influenced by the level of retinol provided in the BFC-1 beta culture medium. BFC-1 beta cells secreted newly synthesized RBP into the culture medium at a rate of 43 +/- 14 ng RBP/24 h/10(6) adipocytes. When the BFC-1 beta adipocytes were provided 1.0 microM retinol in the medium, they accumulated the retinol and synthesized retinyl esters. These studies with BFC-1 beta cells confirm that RBP synthesis and secretion and retinol accumulation are intrinsic properties of differentiated adipocytes. Furthermore, they suggest that RBP and cellular retinol-binding protein gene expression are regulated as part of a package of genes which are modulated during adipocyte differentiation.     

Lipoprotein ApoC-II activation of lipoprotein lipase. Modulation by apolipoprotein A-IV

Lipoprotein lipase (LPL)-mediated hydrolysis of triglycerides (TG) contained in chylomicrons requires the presence of a cofactor, apolipoprotein (apo) C-II. The physiological mechanism by which chylomicrons gain apoC-II necessary for LPL activation in whole plasma is not known. Using a gum arabic stabilized TG emulsion, activation of LPL by lipoprotein apoC-II was studied. Hydrolysis of TG by LPL was greater in the presence of serum than with addition of either high density lipoproteins (HDL) or very low density lipoproteins (VLDL). LPL activation by either VLDL or HDL increased with addition of the lipoprotein-free fraction of plasma. A similar increase in LPL activity by addition of the lipoprotein-free fraction together with HDL or VLDL was observed when another TG emulsion (Intralipid) or TG-rich lipoproteins from an apoC-II deficient subject were used as a substrate. Human apoA-IV, apoA-I, apoE, and cholesteryl ester transfer protein were assessed for their ability to increase LPL activity in the presence of VLDL. At and below physiological concentrations, only apoA-IV increased LPL activity. One hundred percent of LPL activity measured in the presence of serum was achieved using VLDL plus apoA-IV. In the absence of an apoC-II source, apoA-IV had no effect on LPL activity. Removal of greater than 80% of the apoA-IV from the nonlipoprotein-containing fraction of plasma by incubation with Intralipid markedly reduced its ability to activate LPL in the presence of VLDL or HDL. Gel filtration chromatography demonstrated that incubation of the nonlipoprotein-containing fraction of plasma with HDL and the TG emulsion caused increased transfer of apoC-II to the emulsion and association of apoA-IV with HDL. Our studies demonstrate that apoA-IV increases LPL activation in the presence of lipoproteins. We hypothesize that apoA-IV is required for efficient release of apoC-II from either HDL or VLDL, which then allows for LPL-mediated hydrolysis of TG in nascent chylomicrons.     

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.     

Triacylglycerol-rich lipoproteins protect lipoprotein lipase from inactivation by ANGPTL3 and ANGPTL4

Lipoprotein lipase (LPL) is important for clearance of triacylglycerols (TG) from plasma both as an enzyme and as a bridging factor between lipoproteins and receptors for endocytosis. The amount of LPL at the luminal side of the capillary endothelium determines to what extent lipids are taken up. Mechanisms to control both the activity of LPL and its transport to the endothelial sites are regulated, but poorly understood. Angiopoietin-like proteins (ANGPTLs) 3 and 4 are potential control proteins for LPL, but plasma concentrations of ANGPTLs do not correlate with plasma TG levels. We investigated the effects of recombinant human N-terminal (NT) ANGPTLs3 and 4 on LPL-mediated bridging of TG-rich lipoproteins to primary mouse hepatocytes and found that the NT-ANGPTLs, in concentrations sufficient to cause inactivation of LPL in vitro, were unable to prevent LPL-mediated lipoprotein uptake. We therefore investigated the effects of lipoproteins (chylomicrons, VLDL and LDL) on the inactivation of LPL in vitro by NT-ANGPTLs3 and 4 and found that LPL activity was protected by TG-rich lipoproteins. In vivo, postprandial TG protected LPL from inactivation by recombinant NT-ANGPTL4 injected to mice. We conclude that lipoprotein-bound LPL is stabilized against inactivation by ANGPTLs. The levels of ANGPTLs found in blood may not be sufficient to overcome this stabilization. Therefore it is likely that the prime site of action of ANGPTLs on LPL is in subendothelial compartments where TG-rich lipoprotein concentration is lower than in blood. This could explain why the plasma levels of TG and ANGPTLs do not correlate.     

Apolipoprotein E content of VLDL limits LPL-mediated triglyceride hydrolysis

High levels of circulating triglycerides (TGs), or hypertriglyceridemia, are key components of metabolic diseases, such as type 2 diabetes, metabolic syndrome, and CVD. As TGs are carried by lipoproteins in plasma, hypertriglyceridemia can result from overproduction or lack of clearance of TG-rich lipoproteins (TRLs) such as VLDLs. The primary driver of TRL clearance is TG hydrolysis mediated by LPL. LPL is regulated by numerous TRL protein components, including the cofactor apolipoprotein C-II, but it is not clear how their effects combine to impact TRL hydrolysis across individuals. Using a novel assay designed to mimic human plasma conditions in vitro, we tested the ability of VLDL from 15 normolipidemic donors to act as substrates for human LPL. We found a striking 10-fold difference in hydrolysis rates across individuals when the particles were compared on a protein or a TG basis. While VLDL TG contents moderately correlated with hydrolysis rate, we noticed substantial variations in non-apoB proteins within these particles by MS. The ability of LPL to hydrolyze VLDL TGs did not correlate with apolipoprotein C-II content, but it was strongly inversely correlated with apolipoprotein E (APOE) and, to a lesser extent, apolipoprotein A-II. Addition of exogenous APOE inhibited LPL lipolysis in a dose-dependent manner. The APOE3 and (particularly) APOE4 isoforms were effective at limiting LPL hydrolysis, whereas APOE2 was not. We conclude that APOE on VLDL modulates LPL activity and could be a relevant factor in the pathogenesis of metabolic disease.     

Lipolysis products promote the formation of complexes of very-low-density and low-density lipoproteins

In the course of lipolysis, surface lipid products may accumulate on very-low-density lipoproteins (VLDL). To investigate potential lipoprotein interactions mediated by such products, radiolabeled low-density lipoproteins (LDL) were incubated with VLDL and bovine milk lipoprotein lipase in the presence of limited free fatty acid acceptor. With partial VLDL degradation, association of radiolabeled LDL with VLDL remnants or larger aggregates of VLDL density was demonstrated by gradient gel electrophoresis, agarose chromatography, and density gradient ultracentrifugation. VLDL-LDL complex formation was also observed in incubations with lipid extracts from lipolyzed VLDL or with purified palmitic acid in the absence of lipolysis. Complex formation was inhibited by addition of increasing amounts of albumin as free fatty acid acceptor, but could be detected at molar ratios of free fatty acids/albumin that occur in vivo. Composition analysis of LDL reisolated following incubation with VLDL and lipase under conditions favoring partial complex formation revealed enrichment in glycerides and depletion of cholesterol. We conclude that lipolysis products can promote the formation of stable complexes of LDL and VLDL, and that physical interactions of this nature may play a role in the transfer of lipids and apolipoproteins between lipoprotein particles.     

Mechanism for Endogenously Expressed ApoE Modulation of Adipocyte Very Low Density Lipoprotein Metabolism: ROLE IN ENDOCYTIC AND LIPASE-MEDIATED METABOLIC PATHWAYS*

Triglyceride-rich lipoproteins distribute energy in the form of fatty acids to peripheral tissues. We have previously shown that the absence of endogenous adipocyte apoE expression impairs adipocyte triglyceride acquisition from apoE-containing triglyceride-rich lipoproteins in vitro and in vivo. Studies were performed to evaluate the mechanism(s) for this impairment. We excluded a role for secreted apoE in accounting for the difference in very low density lipoprotein (VLDL)-induced adipocyte triglyceride accumulation using cross-incubation studies to show that secreted apoE did not enhance triglyceride synthesis in apoE knockout (EKO) adipocytes incubated with apoE-containing VLDL. Subsequent experiments established that both endocytic and lipase-mediated pathways for lipid acquisition from VLDL were impaired in EKO adipocytes. Binding and internalization of VLDL to EKO adipocytes were significantly lower due to decreased expression or redistribution of low density lipoprotein receptor family proteins. An important role for the VLDL receptor for contributing to differences in VLDL binding between wild-type and EKO adipocytes was identified. Lipoprotein lipase-dependent adipocyte lipogenesis was also significantly decreased in EKO adipocytes even though they secreted as much or more lipolytic activity. This decrease was related to impaired fatty acid internalization in EKO cells. Evaluation of potential mechanisms revealed reduced caveolin-1 and plasma membrane raft expression in EKO adipocytes. Increasing caveolin expression in EKO adipocytes increased fatty acid internalization. Our results establish a role for endogenous adipocyte apoE in VLDL-induced adipocyte lipogenesis by impacting both endocytic and lipoprotein lipase-mediated metabolic pathways. Reduced adipocyte apoE expression, for example that accompanying obesity, will suppress adipocyte acquisition of lipid from apoE-containing VLDL.     

Lipolytic degradation of human very low density lipoproteins by human milk lipoprotein lipase: the identification of lipoprotein B as the main lipoprotein degradation product

Although the direct conversion of very low density lipoproteins (VLDL) into low density (LDL) and high density (HDL) lipoproteins only requires lipoprotein lipase (LPL) as a catalyst and albumin as the fatty acid acceptor, the in vitro-formed LDL and HDL differ chemically from their native counterparts. To investigate the reason(s) for these differences, VLDL were treated with human milk LPL in the presence of albumin, and the LPL-generated LDL1-, LDL2-, and HDL-like particles were characterized by lipid and apolipoprotein composition. Results showed that the removal of apolipoproteins B, C, and E from VLDL was proportional to the degree of triglyceride hydrolysis with LDL2 particles as the major and LDL1 and HDL + VHDL particles as the minor products of a complete in vitro lipolysis of VLDL. In comparison with native counterparts, the in vitro-formed LDL2 and HDL + VHDL were characterized by lower levels of triglyceride and cholesterol ester and higher levels of free cholesterol and lipid phosphorus. The characterization of lipoprotein particles present in the in vitro-produced LDL2 showed that, as in plasma LDL2, lipoprotein B (LP-B) was the major apolipoprotein B-containing lipoprotein accounting for over 90% of the total apolipoprotein B. Other, minor species of apolipoprotein B-containing lipoproteins included LP-B:C-I:E and LP-B:C-I:C-II:C-III. The lipid composition of in vitro-formed LP-B closely resembled that of plasma LP-B. The major parts of apolipoproteins C and E present in VLDL were released to HDL + VHDL as simple, cholesterol/phospholipid-rich lipoproteins including LP-C-I, LP-C-II, LP-C-III, and LP-E. However, some of these same simple lipoprotein particles were present after ultracentrifugation in the LDL2 density segment because of their hydrated density and/or because they formed, in the absence of naturally occurring acceptors (LP-A-I:A-II), weak associations with LP-B. Thus, the presence of varying amounts of these cholesterol/phospholipid-rich lipoproteins in the in vitro-formed LDL2 appears to be the main reason for their compositional difference from native LDL2. These results demonstrate that the formation of LP-B as the major apolipoprotein B-containing product of VLDL lipolysis only requires LPL as a catalyst and albumin as the fatty acid acceptor. However, under physiological circumstances, other modulating agents are necessary to prevent the accumulation and interaction of phospholipid/cholesterol-rich apolipoprotein C- and E-containing particles.     

Mechanisms of enhancement of cholesteryl ester transfer protein activity by lipolysis

Lipoprotein lipase enhances the cholesteryl ester transfer protein (CETP)-mediated transfer of cholesteryl esters from plasma high density lipoproteins (HDL) to very low density lipoproteins (VLDL). In time course studies the stimulation of cholesteryl ester transfer by bovine milk lipase was correlated with accumulation of fatty acids in VLDL remnants. As the amount of fatty acid-poor albumin in the incubations was increased, there was decreased accumulation of fatty acids in VLDL remnants and a parallel decrease in the stimulation of cholesteryl ester transfer by lipolysis. Addition of sodium oleate to VLDL and albumin resulted in stimulation of the CETP-mediated transfer of cholesteryl esters from HDL to VLDL. The stimulation of transfer of cholesteryl esters into previously lipolyzed VLDL was abolished by lowering the pH from 7.5 to 6.0, consistent with a role of lipoprotein ionized fatty acids. CETP-mediated cholesteryl ester transfer from HDL to VLDL was also augmented by phosholipase A2 and by a bacterial lipase which lacked phospholipase activity. When VLDL and HDL were re-isolated after a lipolysis experiment, both lipoproteins stimulated CETP activity. Postlipolysis VLDL and HDL bound much more CETP than native VLDL or HDL. Lipolysis of apoprotein-free phospholipid/triglyceride emulsions also resulted in enhanced binding of CETP to the emulsion particles. Incubation conditions which abolished the enhanced cholesteryl ester transfer into VLDL remnants reduced binding of CETP to remnants, emulsions, and HDL. In conclusion, the enhanced CETP-mediated transfer of cholesteryl esters from HDL to VLDL during lipolysis is related to the accumulation of products of lipolysis, especially fatty acids, in the lipoproteins. Lipids accumulating in VLDL remnants and HDL as a result of lipolysis may augment binding of CETP to these lipoproteins, leading to more efficient transfer of cholesteryl esters from HDL to VLDL.     

Apolipoprotein E and lipoprotein lipase secreted from human monocyte-derived macrophages modulate very low density lipoprotein uptake

We investigated the roles of lipoprotein lipase and apolipoprotein E (apoE) secreted from human monocyte-derived macrophages in the uptake of very low density lipoproteins (VLDL). ApoCII-deficient VLDL were isolated from a patient with apoCII deficiency. The lipolytic conversion to higher density and the degradation of the apoCII-deficient VLDL by macrophages were very slight, whereas the addition of apoCII enhanced both their conversion and degradation. This suggests that the lipolysis and subsequent conversion of VLDL to lipoproteins of higher density are essential for the VLDL uptake by macrophages. VLDL incubated with macrophages obtained from subjects with E3/3 phenotype (E3/3-macrophages) showed a 17-fold greater affinity in inhibiting the binding of 2 micrograms/ml 125I-low density lipoprotein (LDL) to fibroblasts than native VLDL, whereas the incubation of VLDL with macrophages obtained from a subject with E2/2 phenotype (E2/2-macrophages) did not cause any increase in their affinity. Furthermore, 3 micrograms/ml 125I-VLDL obtained from a subject with E3/3 phenotype were degraded by E3/3-macrophages to a greater extent than by E2/2-macrophages (2-fold), indicating that VLDL uptake is influenced by the phenotype of apoE secreted by macrophages. From these results, we conclude that both lipolysis by lipoprotein lipase and incorporation of apoE secreted from macrophages alter the affinity of VLDL for the LDL receptors on the cells, resulting in facilitation of their receptor-mediated endocytosis.     

Adipocyte-derived lipoprotein lipase induces macrophage activation and monocyte adhesion: role of fatty acids

Objective: We evaluated the effect of adipocyte‐derived lipoprotein lipase (LPL) on macrophage activation and monocyte adhesion and the role of fatty acids in these effects. Research Methods and Procedures: 3T3‐L1 adipocytes were incubated with heparin or insulin to induce LPL secretion; then adipocyte conditioned media (CM) were added to cultured J774 macrophages or human aortic endothelial cells (HAECs). Macrophage cytokine production and monocyte adhesion to HAECs were determined. Results: Incubation of macrophages with heparin‐ or insulin‐treated adipocyte CM increased tumor necrosis factor α, interleukin‐6, and nitric oxide production by these cells. LPL neutralization and heparinase treatment prevented these effects. Addition of active LPL or palmitate to cultured macrophages replicated these effects. Blockade of leptin also reduced the effect of insulin‐treated adipocyte CM on macrophage inflammatory changes. Induction of macrophage cytokine secretion by leptin was prevented by LPL immunoneutralization. Finally, addition of CM of heparin‐ or insulin‐treated adipocytes to HAECs stimulated monocyte adhesion to these cells, an effect that was abrogated by an anti‐LPL antibody. This effect was reproduced by treating HAECs with active LPL or palmitate. Discussion: These results point to an effect of LPL‐mediated lipolysis in macrophage activation and monocyte adhesion.     

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.     

Transfer of very low density lipoprotein-associated phospholipids to activated human platelets

LDL-associated phospholipids (PLs) may be transferred into platelets. In this work, we characterized the role of VLDLs as PL donors. VLDL transferred radiolabeled PLs to platelets in a temperature- and concentration-dependent manner. LPL stimulated this process through its action on VLDL lipolysis, because it was abolished by tetrahydrolipstatin. LPL also stimulated the platelet production of thromboxane B2 (TXB2). Both LPL actions were inhibited in the presence of fatty acid-free albumin, suggesting that they were attributable to fatty acids generated during VLDL lipolysis. To study the relationship between PL transfers and platelet activation, we performed incubations in the presence of HDL, a physiological acceptor of PL released from VLDL. HDL antagonized the transfer of PL from VLDL to platelets but had no effect on the production of TXB2, suggesting that PL transfers were driven by platelet activation. Confirming this idea, thrombin stimulated both the production of TXB2 and the transfers of PL. In conclusion, VLDL can transfer PL to platelets. These transfers are stimulated by LPL and thrombin through their action on platelet activation. They might be enhanced in pathologies characterized by increased VLDL concentrations.     

Effect of human high density lipoproteins, anti-apolipoproteins CII and CIII, and hydrolysis of very low density lipoprotein (VLDL) cholesterol ester on VLDL catabolism in vitro

Lipolysis of human very low density lipoproteins (VLDL) by lipoprotein lipase (LPL) was inhibited in the presence of high density lipoproteins (HDL), anti-apolipoprotein (apo) CII, and by increasing the VLDL free cholesterol content but not with anti-apo CIII or lipoprotein-free plasma. The experiments lend direct evidence that the composition of VLDL and their milieu are important determinants of lipolysis by LPL. Apo CIII may not be critical in LPL mediated VLDL catabolism.