Biomedical subjects

I J Goldberg

Publications and source records attributed to I J Goldberg.

At least 19 recordsLinked to original sources

PubMed1995

Lipoprotein lipase association with lipoproteins involves protein-protein interaction with apolipoprotein B.

Lipoprotein lipase (LPL) hydrolyzes chylomicron and very low density lipoprotein (VLDL) triglycerides and potentiates the cellular uptake of lipoproteins. These LPL-lipoprotein associations could involve only protein-lipid interaction, or they could be modulated by apolipoproteins (apo). ApoB is the major protein component of chylomicrons, VLDL, and low density lipoprotein (LDL). ApoB100, a large glycoprotein with a molecular mass of 550 kDa, is composed of several functional domains. A carboxyl-terminal region of the protein is the ligand for the LDL receptor. There are several hydrophobic domains that are believed to be important in lipid binding. The relatively hydrophilic amino-terminal region of apoB, however, has no known function. Using solid phase assays we quantified LPL-lipoprotein complex formation. On a molar basis, severalfold greater amounts of LPL bound to LDL and VLDL than to high density lipoprotein at all the concentrations of LPL tested (0.9-55 nM). To assess the roles of LDL protein versus lipid, we performed competition and ligand blotting experiments. LDL and an amino-terminal fragment of apoB competed better for 125I-LPL binding to LDL than did lipid emulsion particles. Delipidation of LDL-coated plates did not alter LPL binding. On ligand blots, LPL bound to amino-terminal fragments of apoB generated by thrombin digestion but not to apoA1, apoE, or carboxyl-terminal fragments of apoB. Further evidence for LPL interaction with the amino-terminal region of apoB was obtained using anti-apoB monoclonal antibodies. Antibodies directed against the amino-terminal regions of apoB blocked LPL interaction with LDL, whereas those against the carboxyl-terminal region of apoB did not inhibit LPL interaction with LDL. Thus, we conclude that a specific interaction between LPL and the amino-terminal region of apoB may facilitate LPL association with circulating lipoproteins.

Antibodies, Monoclonal↗

PubMed1995

Differentiated macrophages synthesize a heparan sulfate proteoglycan and an oversulfated chondroitin sulfate proteoglycan that bind lipoprotein lipase.

Lipoprotein lipase (LpL), which facilitates lipoprotein uptake by macrophages, associates with the cell surface by binding to proteoglycans (PGs). Studies were designed to identify and characterize specific PGs that serve as receptors for LpL and to examine effects of cell differentiation on LpL binding. PG synthesis was examined by radiolabeling THP-1 monocytes and macrophages (a cell line originally derived from a patient with acute monocytic leukemia) with [35S]sodium sulfate and [3H]serine or [3H]glucosamine. Radiolabeled PGs isolated from the cell surface were purified by chromatography and identified as chondroitin-4-sulfate (CS) PG and heparan sulfate (HS) PG. A sixfold increase in CSPG and an 11-fold increase in HSPG accompanied cell differentiation. Whereas HS glycosaminoglycan chains from both monocytes and macrophages were 7.5 kD in size, CS chains increased in size from 17 kD to 36 kD with cell differentiation, and contained hexuronyl N-acetylgalactosamine-4,6-di-O sulfate disaccharides. LpL binding was sevenfold higher to differentiated cells, and affinity chromatography demonstrated that two cell surface PGs bound to LpL: HSPG and the oversulfated CSPG produced only by differentiated cells. We conclude that differentiation-associated changes in cell surface PG of human macrophages have functional consequences that could increase the atherogenic potential of the cells.

Cell Differentiation↗

PubMed1994

Oligosaccharide sequences of endothelial cell surface heparan sulfate proteoglycan with affinity for lipoprotein lipase.

Lipoprotein lipase (LpL) catalyzes the hydrolysis of triglycerides in plasma lipoproteins at the luminal surface of the vascular endothelium. This enzyme is bound via electrostatic interactions to heparan sulfate (HS). The specific endothelial cell surface HS oligosaccharide sequences that are necessary for binding of LpL to HS have not been characterized. To identify this LpL-binding oligosaccharide sequence, oligosaccharides were isolated from bovine aortic endothelial cell-derived HS and assessed for LpL binding properties. Endothelial HS chains that were isolated from endothelial total cell-associated proteoglycans were deacetylated by complete hydrazinolysis, cleaved with nitrous acid (pH 4.5), and reduced with [3H]NaBH4. The resulting fragments composed of N-sulfated glucosamine-rich oligosaccharides terminating with [3H]2,5-anhydromannitol (AManR) were chromatographed on a LpL-Sepharose column. A high affinity decasaccharide was isolated and characterized. Disaccharide analysis of this decasaccharide indicated that it yielded only the disaccharide IdceA(2-SO4)-->AManR(6-SO4) on treatment with nitrous acid at low pH. Therefore, the sequence of the LpL-binding decasaccharide is [IdceA(2-SO4) alpha 1-4GlcNSO4(6-S0(4)) alpha 1-4]4-IdceA(2-SO4) alpha 1-4AManR(6-SO4) and is distinct from those that bind antithrombin and basic fibroblast growth factor. Partial depolymerization of endothelial HS chains with hydrazine/high pH nitrous acid treatment gave rise to lipase-binding oligosaccharides larger than decasaccharide. However, further complete depolymerization of these oligosaccharides resulted in only a high affinity decasaccharide composed of repeating disaccharide units of [IdceA(2-SO4) alpha 1-4GlcNSO4(6-S0(4))]. These results indicate that the decasaccharide is the active fragment that binds to LpL with high affinity. Molecular modeling studies of the decasaccharide indicate that it presents a linear array of negatively charged sulfate groups that may adopt a favorable disposition to bind to peptide region(s) comprised of basic amino acid residues of LpL with high affinity.

Animals↗

PubMed1994

Lipoprotein lipase hydrolysis of retinyl ester. Possible implications for retinoid uptake by cells.

Adipose tissue contains substantial stores of retinoid (retinol+retinyl ester) that, quantitatively, are second only to retinoid stores in the liver. Our studies show that retinoid levels in adipose tissue are markedly influenced by dietary retinoid intake. Because lipoprotein lipase (LPL) increases the uptake of lipoproteins and lipid emulsion particles by many cell types including adipocytes, we investigated whether LPL also increases retinoid uptake by adipocytes from lipid-containing particles. Addition of LPL (10 micrograms/ml) to BFC-1 beta adipocytes produced a 2-fold increase in cellular uptake of [3H]retinoid from a lipid emulsion containing [3H]retinyl ester. Heparin, which displaces LPL from binding sites on cell surface proteoglycans, increased [3H]retinoid uptake by an additional 2-fold. High performance liquid chromatography analyses showed that greater than 75% of the media and 85% of the cellular radioactivity was present as retinol. The conversion of retinyl ester to retinol by LPL was then assessed using model retinyl ester containing lipid emulsions. Although triglyceride appears to be the preferred substrate for LPL, after greater than 25% of the triglyceride was hydrolyzed, significant amounts of retinyl ester were hydrolyzed by LPL. Retinyl ester hydrolysis was increased approximately 20-fold in the presence of a source of apolipoprotein C-II. The physiologically significant palmitate, stearate, oleate, and linoleate esters of retinol were all hydrolyzed by LPL. When LPL was incubated with [3H]retinyl ester containing rabbit mesenteric chylomicrons and in the presence of heparin and apolipoprotein C-II, the LPL was able to completely hydrolyze the retinyl ester to retinol. Thus, LPL is able to catalyze the hydrolysis of retinyl esters and, through the process of hydrolysis, may facilitate uptake of retinoid by adipocytes.

Adipocytes↗

PubMed1994

Cellular differences in lipoprotein lipase-mediated uptake of low density lipoproteins.

Lipoprotein lipase (LPL) increases the cellular uptake and degradation of LDL by fibroblasts and macrophages via a heparin-sensitive process. The roles of the LDL receptor, LDL receptor-related protein (LRP), and proteoglycans in this process were studied. In up-regulated human fibroblasts, LPL increased degradation of 125I-low density lipoprotein (LDL) (5 micrograms/ml) only 30% during a 6-h incubation at 37 degrees C. Monoclonal antibody 47 (which interacts with the receptor binding region of apoB) decreased LDL degradation 93% in the absence of LPL, but did not reduce the LPL-mediated increase in degradation. In contrast, addition of the 39-kDa receptor-associated protein (RAP) caused a 43% decrease in the LPL-dependent LDL degradation in non-up-regulated fibroblasts. Monoclonal antibody 47 did not decrease LDL degradation by THP-1 macrophages and RAP caused a < 13% decrease in LPL-mediated LDL degradation. LPL also increased the association of acetyl LDL with the surface of the macrophages but did not increase acetyl LDL degradation. The kinetics of LPL-mediated LDL metabolism in macrophages was then compared with that in fibroblasts. The half-lives of cell surface LDL and LPL during a subsequent 37 degrees C incubation were approximately 1 h in THP-1 cells versus 6 h in fibroblasts. In addition, 50% of the 125I-LDL and 30% of the 125I-LPL were degraded within 3 h. After metabolic labeling of THP-1 proteoglycans with 35SO4, > 30% of pericellular heparan sulfate was lost between 2-4 h of the chase period. Therefore, some of the LPL-mediated LDL degradation in the THP-1 cells could be accounted for by internalization of cell surface proteoglycans. We conclude that LRP, but not the LDL receptor, is involved in LPL-mediated degradation of LDL in fibroblasts. This process is much more rapid in THP-1 cells and in addition to LRP may involve other receptors and internalization of proteoglycans.

Acetylation↗

PubMed1994

An amino-terminal fragment of apolipoprotein B binds to lipoprotein lipase and may facilitate its binding to endothelial cells.

Lipoprotein lipase (LPL), the principal enzyme which hydrolyzes triglycerides in circulating plasma lipoproteins, functions while bound to the luminal surface of endothelial cells. LPL is a heparin-binding protein and has been assumed to associate with endothelial cell heparan sulfate proteoglycans (HSPG). Recently, using ligand blotting and affinity chromatography we identified a 116-kDa heparin-releasable LPL-binding protein (hrp-116) from endothelial cells which was not a HSPG (Sivaram, P., Klein, M. G., and Goldberg, I. J. (1992) J. Biol. Chem. 267, 16517-16522). This suggested that, like a number of other heparin-binding proteins, LPL binding to cells also involves non-HSPG proteins. Using heparin-agarose affinity chromatography, a 116-kDa LPL-binding protein was purified from endothelial cell extracts. Microsequencing of peptides generated by Lys-C protease digestion revealed complete homology with four different regions in the NH2-terminal part of human apolipoprotein B (apoB). Western blots using anti-apoB monoclonal antibodies (mAb) that recognize the NH2-terminal region of apoB confirmed that a 116-kDa fragment of apoB was present on endothelial cell membranes. Further evidence that LPL associates with the NH2-terminal region of apoB was obtained by showing 1) that an NH2-terminal fragment of apoB obtained from apoB-transfected CHO cells bound LPL on ligand blots and 2) that NH2-terminal fragments of apoB generated by thrombin digestion of low density lipoprotein bind LPL. Evidence that the NH2-terminal region of apoB mediates LPL interaction with endothelial cells was obtained using monoclonal antibodies. mAb3 and mAb19, which recognize epitopes near the NH2 terminus of apoB, inhibited 125I-LPL binding to cells by 60-65%. In contrast, mAb47, which has determinants at the COOH-terminal end of apoB, inhibited LPL binding by only about 10%. The inhibitory effects of mAb3 and mAb19 were abolished following treatment of cells with heparin, which removes the 116-kDa LPL-binding protein. Furthermore, incubation of 125I-LPL in medium containing an NH2-terminal apoB fragment reduced LPL binding to cells. These data suggest that an NH2-terminal fragment of apoB that binds to endothelial surfaces facilitates LPL binding to cells.

Amino Acid Sequence↗

PubMed1994

Endothelial cells and atherosclerosis: lipoprotein metabolism, matrix interactions, and monocyte recruitment.

The endothelial cell and subendothelial cell matrix provide a stage for several interactions that mediate lipoprotein transport, retention, and modification during atherogenesis. Many novel mechanisms in these processes have been uncovered. The endothelium regulates circulating plasma lipoproteins by acting as the site of action of the triglyceride hydrolyzing enzyme, lipoprotein lipase. Endothelial cells synthesize proteoglycans that anchor this enzyme, affect the actions of growth factors, and directly interact with some classes of lipoproteins. Recent studies have provided insights into the initiation of monocyte entry and the regulation of macrophage lipid uptake. Endothelial cell adhesion molecules are required for the recruitment of monocytes and, therefore, are essential to the atherosclerotic process. Understanding cellular and molecular pathways underlying the early stages of atherosclerosis may provide a rational basis for therapeutic intervention of this disease.

Animals↗

PubMed1994

Overexpression of apolipoprotein CII causes hypertriglyceridemia in transgenic mice.

We have generated transgenic mice expressing the human apolipoprotein CII (apoCII) gene under the transcriptional control of the human cytochrome P-450 IA1 (CYPIA1) promoter. Human apoCII transgenic (HuCIITg) mice exhibited significant basal expression of the transgene (plasma apoCII level = 26.1 +/- 4 mg/dl) and showed further induction of transgene expression after treatment with beta-naphthoflavone. Unexpectedly, HuCIITg mice were hypertriglyceridemic and human apoCII levels correlated strongly to triglyceride levels (R = 0.89, P < 0.0001). Triglyceride levels (mg/dl +/- SEM) were elevated compared to controls in both the fed (804 +/- 113 vs 146 +/- 18, P < 0.001) and fasted (273 +/- 39 vs 61 +/- 4, P < 0.001) states. HuCIITg mice accumulated triglyceride-rich very low density lipoproteins (VLDL) with an increased apoC/apoE ratio. Tracer kinetic studies indicated delayed clearance of VLDL-triglyceride, and studies using Triton inhibition of VLDL clearance showed no increase in VLDL production. Plasma from these mice activated mouse lipoprotein lipase normally and radiolabeled VLDL were normally hydrolyzed. However, HuCIITg VLDL showed markedly decreased binding to heparin-Sepharose, suggesting that apoCII-rich, apoE-poor lipoprotein may be less accessible to cell surface lipases or receptors within their glycosaminoglycan matrices. HuCIITg mice are a promising model of hypertriglyceridemia that suggests a more complex role for apoCII in the metabolism of plasma triglycerides.

Animals↗

PubMed1994

Human ApoA-II inhibits the hydrolysis of HDL triglyceride and the decrease of HDL size induced by hypertriglyceridemia and cholesteryl ester transfer protein in transgenic mice.

The plasma cholesteryl ester transfer protein (CETP) mediates the exchange of HDL cholesteryl esters with triglycerides of other lipoproteins. Subsequent lipolysis of the triglyceride-enriched HDL by hepatic lipase leads to reductions of HDL size and apoA-I content. To investigate a possible modulation of the effects of CETP by apoA-II, human CETP transgenic mice were cross-bred with transgenic mice expressing human apoA-II and, in some cases, human apoA-I and apoC-III (with human-like HDL and hypertriglyceridemia). CETP expression resulted in reductions of HDL and increases in VLDL cholesteryl ester in mice expressing human apoA-II, alone or in combination with apoA-I and apoC-III, indicating that apoA-II does not inhibit the cholesteryl ester transfer activity of CETP. However, CETP expression resulted in more prominent increases in HDL triglyceride in mice expressing both apoA-II and CETP, especially in CETP/apoA-II/apoAI-CIII transgenic mice. CETP expression caused dramatic reductions in HDL size and apoA-I content in apoAI-CIII transgenic mice, but not in apoA-II/AI-CIII transgenic mice. HDL prepared from mice of various genotypes showed inhibition of emulsion-based hepatic lipase activity in proportion to the apoA-II/apoA-I ratio of HDL. The presence of human apoA-II also inhibited mouse plasma hepatic lipase activity on HDL triglyceride. Thus, apoA-II does not inhibit the lipid transfer activity of CETP in vivo. However, coexpression of apoA-II with CETP results in HDL particles that are more triglyceride enriched and resistant to reductions in size and apoA-I content, reflecting inhibition of hepatic lipase by apoA-II. The inhibition of HDL remodeling by apoA-II could explain the relatively constant levels of HDL containing both apoA-I and apoA-II in human populations.

Animals↗

PubMed1994

Lipoprotein lipase (LpL) affects low density lipoprotein (LDL) flux through vascular tissue: evidence that LpL increases LDL accumulation in vascular tissue.

A cardinal feature of the atherosclerotic lesion is increased low density lipoprotein (LDL) content of the arterial wall. Such increases in vascular wall LDL could result from either increased flux of circulating LDL across the arterial endothelial barrier or decreased efflux of LDL that has entered the vascular tissue. A number of studies have focused on factors that alter permeability of endothelial cell monolayers and intact blood vessels causing increased LDL influx. In contrast, the current studies were designed to test the hypothesis that lipoprotein lipase (LpL), increases LDL accumulation and decreases LDL efflux from vascular tissue. Frog mesenteric venular microvessels were cannulated and the rates of fluorescently labeled LDL accumulation (N/t) and efflux (T1/2) were measured by quantitative fluorescence microscopy. When the vessels were perfused with a solution containing bovine milk LpL (10(-5) g/ml) and human LDL (protein = 0.68 mg/ml), N/t was > 15x greater than that of control vessels which were perfused with LDL alone. LpL addition did not change albumin permeability, suggesting that increased N/t was not related to changes in vessel permeability. Increased LDL accumulation within the vessel could have resulted from either an increase in LDL influx from the vessel lumen into the vascular tissue or a decrease in efflux of LDL. Therefore, LDL efflux from vascular tissue was determined by measuring the rate of decline in fluorescence intensity of control and LpL-treated vessels after washout of the vessel lumen with a clear, nonfluorescent solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Albumins↗

PubMed1993

Biotinylation of lipoprotein lipase and hepatic triglyceride lipase: application in the assessment of cell binding sites.

Lipoprotein lipase (LPL) and hepatic triglyceride lipase (HL) were biotinylated using N-hydroxysuccinamide ester of biotin (25-fold molar excess) which was incorporated into the lysine amino groups of the enzyme protein. By assessing enzyme activity and heparin-agarose affinity a biotinylation protocol which did not denature lipases was developed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that biotinylated LPL (bLPL) has the same mobility as that of unlabeled or iodinated LPL. Receptor binding activity of bLPL was studied in (i) cell binding experiments using cultured bovine aortic endothelial cells and (ii) ligand blotting experiments using endothelial cell plasma membranes. Endothelial cells in culture bound similar amounts of bLPL and 125I-LPL. We previously described a 116-kDa heparin-releasable LPL binding protein (hrp-116) on endothelial cells. Using biotinylated lipases in ligand blotting experiments we now demonstrate that both bLPL and biotinylated HL can bind to hrp-116. bLPL in addition also bound to low-density lipoprotein receptor related protein in ligand blotting. Thus, our protocol has produced biotinylated lipases which are both chemically and biologically active and can be used instead of iodinated lipases.

Animals↗

PubMed1993

Lipoprotein lipase binding to adipocytes: evidence for the presence of a heparin-sensitive binding protein.

Lipoprotein lipase (LPL) is synthesized by adipocytes, associated with the cell surface, and released from the cells when they are treated with heparin. Release of LPL from the adipocyte is required for LPL to migrate to its physiological site of action on the luminal surface of capillary endothelial cells. To better understand this process, we studied the interaction of LPL with adipocyte cell membrane proteins. With the use of a ligand blot method, LPL specifically bound to a heparin-releasable, 116-kDa protein on mouse-derived brown fat adipose cell (BFC-1 beta) and rat adipocyte membranes. A 116-kDa cell surface protein was metabolically labeled with [35S]methionine and bound to LPL-Sepharose. This suggested that the LPL-binding protein was synthesized by the cells. When BFC-1 beta were treated with heparin to eliminate heparin-sensitive cell surface binding sites, LPL binding to the cells decreased and release of newly synthesized LPL activity increased. 125I-labeled LPL binding to control cells was reduced (> 70%) by a 50-fold excess of unlabeled LPL. The residual LPL binding to heparin-treated cells was, however, not decreased by the addition of unlabeled LPL. These data imply that specific adipocyte surface LPL binding involves heparin-sensitive sites. We hypothesize that the heparin-releasable, 116-kDa LPL-binding protein mediates specific LPL binding to adipocytes and that LPL activity within adipose tissue is regulated, in part, by the interaction of LPL with this binding protein.

Adipocytes↗

PubMed1993

Abnormal activation of lipoprotein lipase by non-equilibrating apoC-II: further evidence for the presence of non-equilibrating pools of apolipoproteins C-II and C-III in plasma lipoproteins.

Using artificial triglyceride emulsions, we have demonstrated the presence of non-equilibrating pools of apolipoproteins C-II and C-III in human plasma lipoproteins. As the concentrations of acceptor triglycerides were increased, a greater fraction of both apoC-II and apoC-III shifted away from the native plasma lipoproteins to the artificial lipid emulsions. All of the apoC-II and apoC-III in very low density and high density lipoproteins (VLDL and HDL), however, could not be removed from native plasma lipoproteins. The percent of total plasma apoC-II and apoC-III that could be recovered in the VLDL and HDL density fractions varied when plasma from different individuals was used. When plasma samples from normotriglyceridemic subjects were used, HDL was the primary donor of apoCs. The percent of total plasma apoCs associated with HDL decreased from 60% to 25% for apoC-II and from 65% to 15% for apoC-III. When plasma samples from hypertriglyceridemic subjects were incubated with artificial lipid emulsions, VLDL was the primary donor of apoCs. HDL from hypertriglyceridemic subjects only accounted for 5-10% of total fasting plasma apoCs and did not contribute significantly to the final apoC contents of the artificial triglyceride emulsions. To evaluate the significance of the depletion of exchangeable apoCs from plasma HDL, we also examined the ability of control and apoC-depleted HDL to serve as activator for bovine milk lipoprotein lipase (LPL) in vitro. When HDL depleted of exchangeable apoCs were used as the source of plasma apolipoproteins for the activation of LPL in vitro, only 5-10% of the maximal activity obtained with native HDL was demonstrated. In fact, in the presence of comparable concentrations of HDL apoC-II, activation of LPL was the least with HDL which lacked exchangeable apoCs. Our data thus indicated that the presence of exchangeable apoC-II on HDL is necessary for the activation of LPL in vitro. This finding is consistent with our data that suggest that HDL from hypertriglyceridemic subjects do not stimulate LPL as well as HDL from normolipidemic subjects.

Animals↗

PubMed1993

Specificity of lipoprotein lipase binding to endothelial cells.

Lipoprotein lipase (LPL) hydrolyzes circulating lipoprotein triglyceride molecules while it is associated with the luminal surface of capillary endothelial cells. The precise molecular mechanism by which LPL attaches to these cells is unknown. LPL and a number of other molecules, including growth factors and clotting factors, bind to heparin-affinity gels and are eluted using high concentrations of salt. Of these molecules, antithrombin III and basic fibroblast growth factor have been shown to bind to specific cell surface heparan sulfate proteoglycans. Recent data from our laboratory (Sivaram et al. 1992. J. Biol. Chem. 267: 16517-16522) have shown that a heparin-sensitive, non-proteoglycan 116-kDa LPL-binding protein is present on cultured bovine aortic endothelial cells (BAEC). A series of experiments was performed to study the specificity of LPL binding to BAEC and to this 116-kDa protein. At low amounts of LPL (1 microgram) 125I-labeled LPL binding to the cells was inhibited up to 82% by the addition of a 20-fold excess of unlabeled LPL. LPL binding to the BAEC was not decreased by the addition of similar amounts of either antithrombin or thrombin. Specific LPL binding was eliminated by incubating the BAEC at 4 degrees C with heparin containing buffer prior to the addition of LPL. Although cellular internalization of 125I-labeled LPL at 37 degrees C was decreased when an excess of each of the three proteins was added to the culture medium, LPL was most effective. Furthermore, when LPL interaction with the 116-kDa binding protein was studied using ligand blots, 125I-labeled LPL binding was blocked only by unlabeled LPL.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Lipoprotein lipase enhances the interaction of low density lipoproteins with artery-derived extracellular matrix proteoglycans.

The association of plasma low density lipoproteins (LDL) with arterial proteoglycans (PG) is of key importance in LDL retention and modification in the artery wall. Lipoprotein lipase (LpL), the rate-limiting enzyme for hydrolysis of lipoprotein triglyceride, is known to bind both LDL and arterial PG. In the presence of LpL, cellular internalization and degradation of LDL is enhanced by a pathway initiated by interaction of LDL with a cell surface heparan sulfate proteoglycan. To determine whether LpL enhances the binding of LDL to arterial chondroitin sulfate (CS)PG and dermatan sulfate (DS)PG, the major extracellular PG of the artery wall, a microtiter plate assay was used to study LpL-PG-LDL interactions. Binding of LDL to both CSPG and DSPG was increased in the presence of LpL but differential effects were seen for the two PG. LpL enhanced the binding of LDL to CSPG a maximum of 20% and to DSPG a maximum of 40%. Heparin displacement of PG binding suggested a greater binding strength for DSPG-LpL-LDL with 0.25 micrograms heparin required to displace 50% of DSPG compared to 0.01 micrograms to displace 50% of CSPG. The greater enhancement of DSPG-LDL interaction by LpL is of particular interest since increases in DSPG correlate with the accumulation of aortic cholesterol. These data suggest that lipoprotein lipase may enhance the interaction of plasma low density lipoprotein with arterial chondroitin sulfate proteoglycan and dermatan sulfate proteoglycan and thus facilitate low density lipoprotein retention in the artery wall.

Animals↗

PubMed1992

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.

Animals↗

PubMed1992

Lipoprotein lipase release from BFC-1 beta adipocytes. Effects of triglyceride-rich lipoproteins and lipolysis products.

Lipoprotein lipase (LPL), synthesized by adipocytes and myocytes, must be transported to the luminal endothelial cell surface where it then interacts with circulating lipoproteins. The first step in this extracellular LPL transport pathway is LPL release from the surface of LPL-synthesizing cells. Because hydrolysis of triglyceride (TG)-rich lipoproteins releases LPL from the apical surface of endothelial cells, we hypothesized that the same substances dissociate LPL from adipocytes. 125I-LPL was bound to the surface of brown adipocytes (BFC-1 beta). LPL binding to the adipocyte surface was greater than to endothelial cell surfaces. Using low concentrations of heparin, more LPL was released from endothelial cells than BFC-1 beta, suggesting that the affinity of LPL binding to the adipocytes was greater than LPL affinity for endothelial cells. Greater than 3-fold more LPL was released from the cell surface when very low density lipoproteins (VLDL) were added to culture medium containing 3% bovine serum albumin. LPL remaining on the cell surface decreased with VLDL addition. Endogenously produced LPL activity was also released from the cells by VLDL. Low and high density lipoproteins did not release 125I-LPL or LPL activity from the adipocytes. To assess whether lipolysis was necessary for LPL release, BFC-1 beta were incubated with TG-rich lipoproteins from a patient with apoCII deficiency. The apoCII-deficient lipoproteins did not release LPL unless an exogenous source of apoCII was added. Apolipoproteins E and Cs and high molar ratios of oleic acid:bovine serum albumin did not release surface-associated LPL. Lysolecithin (25 and 100 microM), but not lecithin, monoglycerides, or diglycerides, released adipocyte surface LPL. Because lysolecithin also released LPL during a 4 degrees C incubation, cellular metabolic functions are not required for LPL dissociation from the cells. Lysolecithin also inhibited LPL binding to endothelial cells; however, this effect was abrogated by addition of bovine serum albumin. We hypothesize that lipolysis products from TG-rich lipoproteins release adipocyte surface LPL, which can then be transported to the luminal endothelial cell surface.

Adipose Tissue, Brown↗

PubMed1992

Effects of intralipid-induced hypertriglyceridemia on plasma high-density lipoprotein metabolism in the cynomolgus monkey.

Low plasma levels of high-density lipoprotein (HDL) and apolipoprotein (apo) A-I often accompany human hypertriglyceridemia. In an animal model of hypertriglyceridemia, the lipoprotein lipase (LPL)-inhibited cynomolgus monkey, we reported that plasma levels of apo A-I were decreased and the fractional catabolic rate (FCR) of HDL apo was increased. To explore whether hypertriglyceridemia alone would alter plasma apo A-I levels and catabolism, hypertriglyceridemia was produced by intravenous (IV) infusion of 20% Intralipid into female cynomolgus monkeys. Baseline plasma triglyceride (TG) levels averaged 106 mg/dL. With infusion of 200 mg/kg/h Intralipid TG, plasma TG levels peaked at 967 mg/dL (range, 413 to 1,069; n = 6). More prolonged or more severe hypertriglyceridemia caused serious complications in several monkeys. Despite the severe hypertriglyceridemia, HDL TG content, HDL apoproteins, and plasma apo A-I levels did not markedly change, suggesting that very little HDL remodeling had occurred. Kinetic studies of HDL protein and apo A-I were performed in four pairs of monkeys. The two tracers were removed from the plasma at identical rates. In five pairs of animals, apo A-I turnover during control and Intralipid-induced hypertriglyceridemia was not significantly different. We hypothesize that apo A-I FCR is a function of HDL composition. Because Intralipid infusion did not alter HDL composition to the same degree as did LPL inhibition, its effects on HDL apo catabolism were not apparent.

Animals↗