Effect of chlorate on the sulfation of lipoprotein lipase and heparan sulfate proteoglycans. Sulfation of heparan sulfate proteoglycans affects lipoprotein lipase degradation

In avian-cultured adipocytes 76% of the newly synthesized lipoprotein lipase is degraded before release into the medium (Cupp, M., Bensadoun, A., and Melford, K. (1987) J. Biol. Chem. 262, 6383-6388). The same group (Cisar, L. A., Hoogewerf, A. J., Cupp, M., Rapport, C. A., and Bensadoun, A. (1989) J. Biol. Chem. 264, 1767-1774) has proposed that the interaction of lipoprotein lipase with a class of cell surface heparan sulfate proteoglycans is necessary for degradation to occur. To test further this hypothesis, the binding capacity of the plasma membrane for the lipase was decreased by inhibiting the sulfation of glycosaminoglycans with sodium chlorate, an inhibitor of sulfate adenyltransferase. Chlorate decreased sulfate incorporation into trypsin-releasable heparan sulfate proteoglycans to 20% of control levels. The amount of uronic acid in the trypsin-releasable heparan sulfate proteoglycans remained constant. Therefore, chlorate decreased sulfation density on heparan sulfate chains by approximately 5-fold. In the same fractions, chlorate increased the median heparan sulfate Mr measured on Sephacryl S-300. Chlorate decreased the maximum binding of 125I-lipoprotein lipase to adipocytes by 4-fold, but no significant effects on the affinity constants were observed. Chlorate increased lipoprotein lipase secretion in a dose-dependent relationship up to 30 mM. Utilizing a pulse-chase protocol, it was shown that lipase synthesis in control and chlorate-treated cells was not significantly different and that the increased secretion could be accounted for by a decreased lipoprotein lipase degradation rate. In control cells 77 +/- 11% of the synthesized enzyme was degraded whereas in chlorate-treated cells degradation was reduced to 42 +/- 9% of the synthesized amount. The present study shows that decreased sulfation of heparan sulfate proteoglycans decreases the maximum binding of the lipase for the adipocyte cell surface. Consistent with the model that binding of lipoprotein lipase to cell surface heparan sulfate is required for lipase degradation, degradation is reduced in chlorate-treated cultures. In this report it is also shown that chlorate inhibits lipoprotein lipase sulfation and that desulfation of the enzyme has no effect on its catalytic efficiency or on its binding to cultured adipocytes.     

Interaction of lipoprotein lipase and receptor-associated protein

Receptor-associated protein (RAP) is a recognized chaperone/escort protein for members of the low density lipoprotein receptor family. In this report, we show that RAP binds to lipoprotein lipase (LPL) and may play a role in the maturation of LPL. Binding of highly purified RAP to LPL was demonstrated in vitro by solid phase assays, surface plasmon resonance, and rate zonal centrifugation. The dissociation constant for this interaction measured by the first two techniques ranged between 2.4 and 13 nM, values similar to those reported for the binding of RAP to LRP or gp330. The specificity of the interaction was demonstrated by competition with a panel of LPL monoclonal antibodies. Rate zonal centrifugation demonstrated the presence of a stable complex with an apparent Mr consistent with the formation of a complex between monomeric LPL and RAP. RAP x LPL complexes were co-immunoprecipitated in adipocyte lysates or from solutions of purified LPL and RAP. The interaction was also demonstrated in whole cells by cross-linking experiments. RAP-deficient adipocytes secreted LPL with a specific activity 2.5-fold lower than the lipase secreted by control cells. Heparin addition to cultured RAP-deficient adipocytes failed to stimulate LPL secretion in the medium, suggesting defective binding of the lipase to the plasma membrane. These studies demonstrate that RAP binds to LPL with high affinity both in purified systems and cell extracts and that RAP-deficient adipocytes secrete poorly assembled LPL. A function of RAP may be to prevent premature interaction of LPL with binding partners in the secretory pathway, namely LRP and heparan sulfate proteoglycan.     

Sortilin/neurotensin receptor-3 binds and mediates degradation of lipoprotein lipase

Lipoprotein lipase and the receptor-associated protein (RAP) bind to overlapping sites on the low density lipoprotein receptor-related protein/alpha2-macroglobulin receptor (LRP). We have investigated if lipoprotein lipase interacts with the RAP binding but structurally distinct receptor sortilin/neurotensin receptor-3. We show, by chemical cross-linking and surface plasmon resonance analysis, that soluble sortilin binds lipoprotein lipase with an affinity similar to that of LRP. The binding was inhibited by heparin and RAP and by the newly discovered sortilin ligand neurotensin. In 35S-labeled 3T3-L1 adipocytes treated with the cross-linker dithiobis(succinimidyl propionate), lipoprotein lipase-containing complexes were isolated by anti-sortilin antibodies. To elucidate function in cells, sortilin-negative Chinese hamster ovary cells were transfected with full-length sortilin and shown to express about 8% of the receptors on the cell surface. These cells degraded 125I-labeled lipoprotein lipase much faster than the wild-type cells. The degradation was inhibited by unlabeled lipoprotein lipase, indicating a saturable pathway, and by RAP and heparin. Moreover, inhibition by the weak base chloroquine suggested that degradation occurs in an acidic vesicle compartment. The results demonstrate that sortilin is a multifunctional receptor that binds lipoprotein lipase and, when expressed on the cell surface, mediates its endocytosis and degradation.     

Heparin decreases the degradation rate of lipoprotein lipase in adipocytes

The mechanism responsible for the stimulation of secretion of lipoprotein lipase by heparin in cultured cells was studied with avian adipocytes in culture. Immunoprecipitation followed by electrophoresis and fluorography were used to isolate and quantitate the radiolabeled enzyme, whereas total lipoprotein lipase was quantitated by radioimmunoassay. Rates of synthesis of lipoprotein lipase were not different for control or heparin treatments as judged by incorporation of L-[35S]methionine counts into lipoprotein lipase during a 20-min pulse. This observation was corroborated in pulse-chase experiments where the calculation of total lipoprotein lipase synthesis, based on the rate of change in enzyme-specific activity during the chase, showed no difference between control (8.13 +/- 3.1) and heparin treatments (9.1 +/- 5.3 ng/h/60-mm dish). Secretion rates of enzyme were calculated from measurements of the radioactivity of the secreted enzyme and the cellular enzyme-specific activity. Degradation rates were calculated by difference between synthesis and secretion rates of enzyme. In control cells 76% of the synthesized enzyme was degraded. Addition of heparin to the culture medium reduced the degradation rate to 21% of the synthetic rate. The presence of heparin in cell media resulted in a decrease in apparent intracellular retention half-time for secreted enzyme from 160 +/- 44 min to 25 +/- 1 min. The above data demonstrate that the increase in lipoprotein lipase protein secretion, observed upon addition of heparin to cultured adipocytes, is due to a decreased degradation rate with no change in synthetic rate. Finally, newly synthesized lipoprotein lipase in cultured adipocytes is secreted constitutively and there is no evidence that it is stored in an intracellular pool.     

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.     

Regulation of lipoprotein lipase. Induction by insulin.

Lipoprotein lipase activity in intact epididymal adipose tissue of fasted rats increased rapidly after treatment with insulin in vivo. In contrast, lipoprotein lipase activity in adipocytes isolated from the contralateral fat pads remained essentially unchanged. When adipocytes were incubated for 30 min at ambient temperature in vitro, about 2 times more lipoprotein lipase activity was found in the medium of cells from insulin-treated rats than in medium from cells of control animals. Following insulin treatment, extracts of tissue acetone powders separated by gel chromatography showed increases in both enzyme activity fractions obtained (designated lipoprotein lipase a and b). However, no consistent differences were observed between fractions derived from adipocyte acetone powders of insulin-treated and control animals. All the observed effects of insulin on lipoprotein lipase activity were abolished by cycloheximide treatment in vivo. These data indicate that following insulin treatment, increased lipoprotein lipase activity in adipose tissue results from enhanced enzyme secretion by the fat cell and subsequent accumulation in the tissue, thus implicating the adipocyte secretory mechanism as a major site of regulation of lipoprotein lipase activity in adipose tissue.     

Fate of lipoprotein lipase taken up by the rat liver. Evidence for a conformational change with loss of catalytic activity

When isolated rat livers were perfused with medium containing lipoprotein lipase, 40-60% was taken up during a single passage. This value was similar for lipoprotein lipase derived from culture medium of rat preadipocytes, and for lipoprotein lipase purified from bovine milk. It was also, similar, irrespective of the lipoprotein lipase concentration, at least up to 1 microgram/ml. Immediately following its uptake by the liver, a large fraction of the lipoprotein lipase could be released by heparin, but the magnitude of this fraction decreased with time. The enzyme lost its catalytic activity rather rapidly, but its degradation to acid-soluble products, or to larger fragments, was much slower. On heparin-agarose chromatography, the enzyme taken up by the liver eluted at a lower salt concentration than the original lipoprotein lipase preparation. This change in affinity for heparin suggests that the originally dimeric lipoprotein lipase had dissociated into monomers, in analogy to the findings in model experiments. It is suggested that the initial uptake of lipoprotein lipase occurs by binding to a polyanion at the liver cell surface. This is followed by endocytosis and dissociation of the enzyme from its heparan sulfate-like binding site. Acidification of the endosome may cause a conformational change in the lipase molecule with dissociation to inactive monomers, preceding ultimate proteolytic degradation.     

Insulin promotes shedding of syndecan ectodomains from 3T3-L1 adipocytes: a proposed mechanism for stabilization of extracellular lipoprotein lipase

Syndecans are a family of four transmembrane heparan sulfate proteoglycans that act as coreceptors for a variety of cell-surface ligands and receptors. Receptor activation in several cell types leads to shedding of syndecan-1 and syndecan-4 ectodomains into the extracellular space by metalloproteinase-mediated cleavage of the syndecan core protein. We have found that 3T3-L1 adipocytes express syndecan-1 and syndecan-4 and that their ectodomains are shed in response to insulin in a dose-, time-, and metalloproteinase-dependent manner. Insulin responsive shedding is not seen in 3T3-L1 fibroblasts. This shedding involves both Ras-MAP kinase and phosphatidylinositol 3-kinase pathways. In response to insulin, adipocytes are known to secrete active lipoprotein lipase, an enzyme that binds to heparan sulfate on the luminal surface of capillary endothelia. Lipoprotein lipase is transported as a stable enzyme from its site of synthesis to its site of action, but the transport mechanism is unknown. Our studies indicate that shed adipocyte syndecans associate with lipoprotein lipase. The shed syndecan ectodomain can stabilize active lipoprotein lipase. These data suggest that syndecan ectodomains, shed by adipocytes in response to insulin, are physiological extracellular chaperones for lipoprotein lipase as it translocates from its site of synthesis to its site of action.     

Intra- and extracellular forms of lipoprotein lipase in adipose tissue.

The location of lipoprotein lipase activity in rat adipose tissue was studied using intact epididymal fat pads, isolated adipocytes, and lipoprotein lipase activity secreted from adipocytes as enzyme sources. The enzyme activities of these preparations were characterized by gel filtration. The method used for isolation of adipocytes had been modified to minimize activation of lipoprotein lipase during the procedures. Extracts of intact adipose tissue separated into two major lipoprotein lipase activity peaks, designated "a" and "b", the "a" fraction representing about 30 (fasted rats) to 50% (fed rats) of the total enzyme activity. An intermediate fraction (designated "i") was frequently observed. Extracts of isolated adipocytes from fed rats contained about 35% and those from fasted rats about 65% of the lipoprotein lipase activity present in intact tissue. The "b" fraction constituted 80--97% of the adipocyte lipoprotein lipase activity. In contrast, the enzyme activity secreted from the adipocytes contained only the "a" and "i" fractions. These data implicate the existance of one intracellular form of lipoprotein lipase (corresponding to the "b" fraction), different from extracellular forms of the enzyme (corresponding to fractions "a" and "i"). A transformation of the intracellular to the extracellular forms appears to occur in conjunction with secretion of enzyme from the fat cell.     

The lipoprotein lipase of white adipose tissue. Changes in the adipocyte cell-surface content of enzyme in response to extracellular effectors in vitro.

An indirect labelled-second-antibody cellular immunoassay for adipocyte surface lipoprotein lipase was used to assess the changes that occurred during the incubation of cells in the presence and absence of effectors. In the absence of any specific effectors, the amount of immunodetectable lipoprotein lipase present at the surface of adipocytes remained constant throughout the 4 h incubation period at 37 degrees C. Under such conditions total cellular enzyme activity also remained constant, with no activity appearing in the medium. In the presence of heparin, cell-surface immunodetectable lipoprotein lipase increased by up to 20%, whereas in the presence of cycloheximide they decreased by up to 60%. Thus the obvious turnover of enzyme from this cell-surface site was found to be relatively rapid and dependent for its replenishment, at least in part, on protein synthesis. In the presence of insulin alone, a substantial increase in cell-surface lipoprotein lipase protein occurred, only part of which was dependent on protein synthesis. The total cellular activity of lipoprotein lipase was unaffected by the presence of insulin. The insulin-dependent increase in cell-surface enzyme was potentiated somewhat in the presence of dexamethasone, which was not shown to exert any independent effect. Glucagon, adrenaline and theophylline all produced a significant decline in the cell-surface immunodetectable lipoprotein lipase, which in the case examined (adrenaline) was partially additive with regard to the independent effect of cycloheximide. Cell-surface immunodetectable lipoprotein lipase amounts were decreased significantly when cells were incubated in the presence of either colchicine or tunicamycin. The concerted way in which cell-surface lipoprotein lipase altered during the incubations of adipocytes in the presence of effectors suggested that the translocation of enzyme to and from this cellular site was dependent on hormonal action and the integrity of intracellular protein-transport mechanisms.     

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.     

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.     

Influence of nutritional state on lipoprotein lipase activities in the hypothyroid rat

We have investigated the effects of nutritional state on the lipoprotein lipase activities of the experimentally hypothyroid rat. Both short-term effects (i.e., those of a 24 h fast with and without re-feeding) and long-term effects (due to decreased food intake in hypothyroidism) have been studied. The hypothyroid rats had significantly higher lipoprotein lipase activities of adipose tissue and heart muscle. The effect of hypothyroidism on adipose tissue lipoprotein lipase activities was modified by the nutritional state. In rats studied after 24 h fasting, the hypothyroid group had significantly higher lipoprotein lipase activities than weight-matched, age-matched and pair-fed (i.e., semi-starved) control groups. In rats studied in the re-fed state, the effects of hypothyroidism as such were less evident, since the pair-fed group also demonstrated significantly higher enzyme activities than did the other control groups. We have also studied the lipoprotein lipase activities of different enzyme preparations from adipose tissue. The effects of hypothyroidism were most clearly reflected in an increase of heparin-elutable enzyme activity from adipose tissue, whereas adipocyte lipoprotein lipase activity and the lipoprotein lipase secretion rate from adipocytes were affected to a lesser extent. We conclude that alterations in food intake strongly influence the lipoprotein lipase activities in the hypothyroidism. Our data also imply that the increased lipoprotein lipase activity in the hypothyroid state is due to a decreased degradation of the enzyme, both intra- and extracellularly.     

Synthesis and secretion of lipoprotein lipase in 3T3-L1 adipocytes. Demonstration of inactive forms of lipase in cells

3T3-L1 adipocytes in culture incorporated [35S]methionine into a protein which could be immunoprecipitated with chicken antiserum to bovine lipoprotein lipase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed this protein had an Mr of 55,000, similar to that of bovine lipoprotein lipase, and accounted for 0.1-0.5% of total protein synthesis in the adipocytes. Lipoprotein lipase protein was present in small amounts in confluent 3T3-L1 fibroblasts, and the amount increased many-fold as the cells differentiated into adipocytes. This increase was accompanied by parallel increases in cellular lipase activity and secretion. When cells were grown with [35S]methionine, the amount of label incorporated into lipoprotein lipase increased for 2 h and then leveled off. Pulse-chase experiments showed that half-life of newly synthesized lipase was about 1 h. Turnover of lipoprotein lipase in control cells involved both release to the medium and intracellular degradation. When N-linked glycosylation was blocked by tunicamycin, the cells synthesized a form of lipase that had a smaller Mr (48,000), was catalytically inactive, and was not released to the medium. Radioimmunoassay demonstrated that 3T3-L1 adipocytes contained an unexpectedly large amount of lipoprotein lipase protein. 55% of the enzyme protein in acetone/ether powder of the cells was insoluble in 50 mM NH3/NH4Cl at pH 8.1, a solution commonly used to extract lipoprotein lipase; 27% of the lipase protein was soluble but did not bind to heparin-Sepharose and had very low lipase activity; and the remaining 13% was soluble, bound to heparin-Sepharose, and had high lipolytic activity. About one-half of the lipase released spontaneously to the medium was inactive, and lipase inactivation proceeded in the medium with little loss of enzyme protein. Lipoprotein lipase released heparin, in contrast, was fully active and more stable. When protein synthesis was blocked by cycloheximide, the level of lipoprotein lipase activity in adipocytes decreased more rapidly than the amount of lipase protein in the cells. Most of the inactive lipoprotein lipase in adipocytes probably results from dissociation of active dimeric lipase, but some could be a precursor of active enzyme.     

A comparison of lipoprotein lipase activity and adipocyte differentiation in growing male rats.

During adipose tissue development changes in lipoprotein lipase activity per adipocyte precede significant changes in fat cell size. Lipoprotein lipase activity per adipocyte increases fourfold from the second to seventh postnatal week. Furthermore, when isolated adipocytes and stromal--vascular cells are prepared by collagenase digestion of adipose tissue, there is a progressive shift in enzyme activity during development from the stromal-vascular compartment to the adipocyte fraction. The data support the concept that during normal development a "bed" of preadipocytes is synthesized during the suckling period. The data further suggest a regulatory role for lipoprotein lipase in the control of "lipid-filling" during early postnatal development.     

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.     

Induction of low density lipoprotein receptor synthesis by high density lipoprotein in cultures of human skin fibroblasts.

Further studies have been made of the effects of high density lipoprotein (HDL) on the surface binding, internalization and degradation of 125I-labeled low density lipoprotein (125I-labeled LDL) by cultured normal human fibroblasts. In agreement with earlier studies, during short incubations HDL inhibited the surface binding of 125I-labeled LDL. In contrast, following prolonged incubations 125I-labeled LDL binding was consistently greater in the presence of HDL. The increment in 125I-labeled LDL binding induced by HDL was: (a) associated with a decrease in cell cholesterol content; (b) inhibited by the addition of cholesterol or cycloheximide to the incubation medium; and (c) accompanied by similar increments in 125I-labeled LDL internalization and degradation. It is concluded that HDL induces the synthesis of high affinity LDL receptors in human fibroblasts by promoting the efflux of cholesterol from the cells.     

The lipoprotein lipase of white adipose tissue. Studies on the intracellular distribution of the adipocyte-associated enzyme.

The separation of rat epididymal adipocytes into plasma-membrane, mitochondrial, microsomal and cytosol fractions is described. The fractions, which were characterized by marker-enzyme analysis and electron-micrographic observation, from the cells of fed and 24 h-starved animals were used to prepare acetone/diethyl ether-dried powders for the measurement of lipoprotein lipase activities. The highest specific activities and proportion of recovered lipoprotein lipase activity were found in the plasma-membrane and microsomal fractions. The two fractions from the cells of fed rats showed similar activities and enrichments of the enzyme, these activities being higher than the plasma-membrane and lower than the microsomal activities recovered from the cells of starved animals. Chicken and guinea-pig anti-(rat lipoprotein lipase) sera were prepared, and an indirect labelled-second-antibody cellular immunoassay, using 125I-labelled rabbit anti-(chicken IgG) or 125I-labelled sheep anti-(guinea-pig IgG) antibodies respectively, for the detection of cell-surface enzyme was devised and optimized. The amount of immunodetectable cell-surface lipoprotein lipase was higher for cells isolated from fed animals than for cells from 24 h-starved animals, when either anti-(lipoprotein lipase) serum was used in the assay. The amount of immunodetectable cell-surface lipoprotein lipase fell further when starvation was extended to 48 h. The lipoprotein lipase of plasma-membrane vesicles was shown to be a patent activity and to be immunodetectable in a modification of the cellular immunoassay. Although the functional significance of the adipocyte surface lipoprotein lipase is not known, the possibility of it forming a pool of enzyme en route to the capillary endothelium is advanced.     

Endogenously produced endothelial lipase enhances binding and cellular processing of plasma lipoproteins via heparan sulfate proteoglycan-mediated pathway

Endothelial lipase (EL) is a new member of the triglyceride lipase gene family, which includes lipoprotein lipase (LpL) and hepatic lipase (HL). Enzymatic activity of EL has been studied before. Here we characterized the ability of EL to bridge lipoproteins to the cell surface. Expression of EL in wild-type Chinese hamster ovary (CHO)-K1 but not in heparan sulfate proteoglycan (HSPG)-deficient CHO-677 cells resulted in 3-4.4-fold increases of 125I-low density lipoprotein (LDL) and 125I-high density lipoprotein 3 binding (HDL3). Inhibition of proteoglycan sulfation by sodium chlorate or incubation of cells with labeled lipoproteins in the presence of heparin (100 microg/ml) abolished bridging effects of EL. An enzymatically inactive EL, EL-S149A, was equally effective in facilitating lipoprotein bridging as native EL. Processing of LDL and HDL differed notably after initial binding via EL to the cell surface. More than 90% of the surface-bound 125I-LDL was destined for internalization and degradation, whereas about 70% of the surface-bound 125I-HDL3 was released back into the medium. These differences were significantly attenuated after HDL clustering was promoted using antibody against apolipoprotein A-I. At equal protein concentration of added lipoproteins the ratio of HDL3 to VLDL bridging via EL was 0.092 compared with 0.174 via HL and 0.002 via LpL. In summary, EL mediates binding and uptake of plasma lipoproteins via a process that is independent of its enzymatic activity, requires cellular heparan sulfate proteoglycans, and is regulated by ligand clustering.     

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.