Endothelium,the dynamic interface in cardiac lipid transport

Vascular endothelium is the dynamic interface in transport of lipid from blood to myocytes in heart and arteries. The luminal surface of endothelium is the site of action of lipoprotein lipase on chylomicrons and VLDL and the site of uptake of fatty acids from albumin. Fatty acids and monoacylglycerols are transported from the lumen in an interfacial continuum of endothelial and myocyte membranes. Lipoprotein lipase is transferred from myocytes to the vascular lumen, and is anchored there, by proteoheparan sulfate in cell membranes. Insulin, needed for synthesis of lipoprotein lipase and esterfication of fatty acids, is captured from the blood stream and delivered to myocytes by endothelial insulin receptors. Fatty acids, monoacylglycerols, lipoprotein lipase and insulin are transported along the same route, but by different mechanisms. The route involves the plasma membrane of endothelium and myocytes, the membrane lining transendothelial channels, and intercellular contacts. (Mol Cell Biochem116: 181–191, 1992)     

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.     

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.     

Hyperlipidemia in mast cell-deficient W/WV mice

Approximately 70% of the W/WV mice lacking mast cells due to a genetic defect showed hypertriglyceridemia combined with hypercholesterolemia. Increases of various magnitudes in chylomicrons, very-low-density lipoprotein, and intermediate-density lipoprotein were observed in the plasma of W/WV mice compared to those in the plasma of congenic normal mice. The increase in these lipoproteins was seen even in normolipidemic W/WV mice. Activities of both lipoprotein lipase and hepatic triacylglycerol lipase in the plasma after heparin injection were markedly lower in the W/WV mice than in the congenic normal mice, although activities of both lipoprotein lipase in the heart and adipose tissue and hepatic triacylglycerol lipase in the liver were not decreased. These results suggest that the W/WV mice have genetic defects in one or more of the following: secretion of both lipases from their synthesising cells, transport to the endothelium, and anchoring to the endothelial surface. Heparin deficiency in these mice may be responsible for the impairment and, thereby, may partially contribute to the hyperlipidemia.     

Immunocytochemical localization of the functional fraction of lipoprotein lipase in the perfused heart

The functional (heparin-releasable) fraction of myocardial lipoprotein lipase (LPL) has been located at the lumen surface of capillary endothelium by means of an indirect immunocytochemical perfusion method for electron microscopy. The primary step immunoreactant was an IgG fraction of goat antiserum directed against LPL from rat heart. The second step antibody, conjugated with horseradish peroxidase, was rabbit IgG directed against goat IgG. Peroxidase reaction product, when present, appeared at the surface an in invaginations of the lumenal plasma membrane of capillary endothelium and also on chylomicrons adherent to that membrane. The highest coverage by such product occurred when the highest heparin-releasable heart LPL activity was attained after fat-feeding of rats. Coverage was low when a low level of heparin-releasable heart LPL activity was induced by carbohydrate-feeding. Coverage was very low in the perfused hearts after heparin-release of functional LPL activity. The positive association between these immunocytochemical results and actual levels of functional LPL activities indicates that functional LPL in the isolated rat heart is at the lumen surface of capillary endothelium.     

Effects of threonine-poor apolipoproteins on post-heparin plasma hepatic triacylglycerol lipase and lipoprotein lipase

Whole-irradiated rabbit pre-heparin plasma had an important inhibitory effect on hepatic triacylglycerol lipase and lipoprotein lipase activities, whereas control rabbit pre-heparin plasma slightly inhibited hepatic triacylglycerol lipase activity at a high concentration and enhanced lipoprotein lipase activity. As some apolipoproteins were known to modulate these two lipolytic enzymes, the inhibitory effects of irradiated rabbit plasma were investigated in apolipoproteins. Three apolipoproteins, with isoelectric points of about 6.58, 6.44 and 6.12, characterized by their low content in threonine (threonine-poor apolipoproteins) were produced in high concentrations in rabbit VLDL and HDL after irradiation. The effects of these apolipoproteins on control rabbit post-heparin plasma hepatic triacylglycerol lipase and extrahepatic lipoprotein lipase were studied. Threonine-poor apolipoproteins substantially inhibited the hepatic triacylglycerol lipase activity and enhanced the apolipoprotein C-II-stimulated activity of lipoprotein lipase. The amounts of these apolipoproteins in triacylglycerol-rich lipoprotein particles may determine the lipolytic activity of lipoprotein lipase and hepatic triacylglycerol lipase in triacylglycerol hydrolysis. The existence of another inhibitor of lipoprotein lipase remains to be determined.     

Possible role of lipoprotein lipase in the regulation of endogenous triacylglycerols in the rat heart.

1. Adrenaline has a biphasic effect on intracellular lipoprotein lipase activity and on endogenous triacylglycerol content in heparin-perfused heart. 2. A high concentration of adrenaline (1 microM in the perfusion buffer) activated endogenous lipoprotein lipase activity and, at the same time, decreased intracellular triacylglycerol stores. 3. In contrast, a low concentration (0.005 microM-adrenaline) inhibited intracellular lipoprotein lipase activity. Under these conditions, cardiac triacylglycerol content was elevated above control values. 4. Perfusing the heart with high and low concentrations of 3-isobutyl-1-methylxanthine elicited a biphasic effect on endogenous lipoprotein lipase activity and triacylglycerol content similar to that seen with adrenaline treatment. 5. The effect of adrenaline on intracellular lipoprotein lipase activity appears to be mediated by cyclic AMP through protein kinase. 6. A possible role for intracellular lipoprotein lipase in the regulation of endogenous triacylglycerol in rat heart is proposed.     

Regulation of the secretion of lipoprotein lipase by mouse macrophages

The regulation of the secretion of lipoprotein lipase was studied in primary cultures of mouse peritoneal macrophages and in the murine macrophage cell line J774. As previously reported, both cell types secrete a lipase with the characteristics of lipoprotein lipase. Incubation of macrophages with insulin, insulin-like growth factor, and L-thyroxine had no effect on lipoprotein lipase secretion. Incubation with dexamethasone and with several agents which increase intracellular cyclic AMP led to a decrease in lipoprotein lipase secretion by mouse peritoneal macrophages. These results suggest that the hormonal regulation of lipoprotein lipase in macrophages is different from that in adipose tissue and heart muscle. Incubation of the macrophages with heparin caused a marked increase in the secretion of lipoprotein lipase. Short incubations with heparin (5 min) caused a release of the enzyme into the media, while longer incubations caused a 2-8-fold increase in net lipoprotein lipase secretion which was maximal after 2-16 h depending on cell type, and persisted for 24 h. The effect of heparin was dose-dependent and specific (it was not duplicated by other glycosaminoglycans). The mechanism of heparin-induced increase in lipoprotein lipase secretion was explored. The increase was not caused by the release of a presynthesized intracellular pool of lipoprotein lipase or by the stabilization of lipoprotein lipase by heparin after secretion. The heparin-induced increase in lipoprotein lipase secretion was dependent on protein synthesis. The secretion of lipoprotein lipase by macrophages in response to low levels of heparin may be a significant factor in the formation of atherosclerotic lesions.     

Lipoprotein lipase activity in the flight muscle of Locusta migratoria and its specificity for haemolymph lipoproteins

Lipoprotein lipase activity in flight muscle homogenates of Locusta migratoria was measured, using natural radiolabelled lipoproteins as substrates. The flight specific lipoprotein A⁺ (or low density lipophorin) stimulated lipoprotein lipase activity several-fold compared to the resting lipoprotein A_y (or high density lipophorin). However, with the high mol. wt lipoprotein fraction O_AKH as a substrate, lipase activity was even doubled compared to lipoprotein A⁺. Lipase activity was not increased in flight muscle homogenates of insects which had flown. Neither adipokinetic hormone, nor octopamine had any direct effect on lipoprotein lipase activity. Aspects of hormonal regulation and apoprotein activation of the locust flight muscle lipoprotein lipase are discussed and compared with the model for vertebrate lipoprotein lipase.     

Lipoprotein lipase: evidence for high- and low-affinity enzyme sites.

The kinetic constants for membrane-supported lipoprotein lipase have been determined for the enzyme active in lipoprotein triglyceride catabolism in perfused heart and adipose tissues, using a nonrecirculating system. Heart endothelial lipoprotein lipase reacted as a single population of high-affinity substrate binding sites (Km' 0.07 mM triglyceride). Km' (apparent Michaelis constant for the supported enzyme species) was independent of flow rate and the enzyme was rapidly released by heparin, suggestive of a superficial membrane binding site. Lipoprotein lipase active in perfused adipose tissue had significantly different kinetic properties, including a low substrate affinity (Km' 0.70 mM triglyceride), diffusion dependence of Km' at low flow rates, and slow release of enzyme by heparin. Adipose tissue may contain a small proportion of high affinity sites. While only a small proportion of total heart tissue lipoprotein lipase was directly active in triglyceride hydrolysis, this study suggests that the major part of lipoprotein lipase in adipose tissue may be involved in the hydrolysis of circulating lipoprotein triglyceride.     

A selective deficiency of hepatic triacylglycerol lipase in guinea pigs

The properties of postheparin plasma triacylglycerol-hydrolyzing enzymes were investigated in guinea pig and rat. In rat, lipoprotein lipase and hepatic triacylglycerol lipase were separated on a heparin-Sepharose affinity chromatography. In postheparin plasma of guinea pig, however, hepatic triacylglycerol lipase was almost completely absent, while lipoprotein lipase was present. Hepatic triacylglycerol lipase was also deficient in the liver tissue extract of guinea pig. Plasma lipoprotein compositions of high-fat fed and control guinea pigs were analyzed. One of the outstanding changes found in high-fat fed animals was the presence of chylomicronemia. One guinea pig showed gross hyperlipemia with triacylglycerol concentrations of 2715 mg/100 ml. Plasma triacylglycerol concentrations of each lipoprotein fraction of very low density, intermediate density, low density and high density lipoproteins from high-fat fed animals were almost the same as those of the corresponding lipoprotein fractions from controls. Discussion was focused on the development of chylomicronemia in relation to the defects of triacylglycerol-hydrolyzing enzyme systems in this animal.     

The effects of oral agent or insulin treatments on the plasma lipoproteins and the plasma lipoprotein lipase activator in diabetic patients

The structure and the metabolism of plasma lipoproteins are altered in diabetes mellitus. Insulin or oral agent treatments affect the lipoprotein metabolism in addition to improving hyperglycemia. However, it is not clear whether the alterations seen in lipoproteins during treatment are related to the degree of diabetic control or to the mode of diabetic treatment. The effects of insulin or oral agent treatments on the plasma lipoproteins and lipoprotein lipase activator were compared in a strictly defined non-obese, non-insulin dependent diabetic patient. Both treatment groups had similar plasma triglyceride, total cholesterol, low and high density lipoprotein cholesterol, and lipoprotein lipase activator levels. Lipoprotein lipase activator contents of the very low density lipoproteins correlated positively with their triglyceride (r = 0.803 in insulin, r = 0.828 in oral agent treated patients) and protein (r = 0.713 in insulin, r = 0.862 in oral agent treated patients) contents. The findings of this study indicated that plasma lipid levels, very low density lipoprotein compositions, and lipoprotein lipase activator contents were not significantly different in non-obese, non-insulin dependent diabetic patients treated with either oral hypoglycemic agents or insulin.     

Apparent microviscosity of intact and post-lipolysis ("remnant") very low density lipoprotein particles.

The apparent microviscosity of intact rat plasma very low density lipoprotein (VLDL) and post-lipolysis very low density lipoprotein was determined by fluorescence depolarization measurements and flurorescence decay measurements using 1, 6-diphenylhexatriene. Post-lipolysis very low density lipoprotein was prepared in vitro after incubation of the intact lipoprotein with either purified bovine milk lipoprotein lipase or lipoprotein lipase rich (post-heparin) plasma. During lipolysis, an average of 88% of the triglycerides were hydrolyzed, and the lipoprotein became depleted in phospholipids, cholesterol and apolipoprotein C. The apparent microviscosity of the lipoprotein increased by three-fold from 0.63 to 1.88 poise. It is concluded that the compositional changes occurring during lipolysis affect the physical properties of the lipoprotein, as measured here by the fluidity (microviscosity) of the particles.     

Effect of diabetes on acid and neutral triacylglycerol lipase and on lipoprotein lipase activities in isolated myocardial cells from rat heart.

A neutral triacylglycerol lipase activity that is separate and distinct from lipoprotein lipase (LPL) could be measured in homogenates of myocardial cells if protamine sulphate and high concentrations of albumin were included in the assay. This neutral lipase was predominantly particulate, with the highest relative specific activity in microsomal subcellular fractions. The induction of diabetes by the administration of streptozotocin to rats resulted in a decrease in LPL activity in myocyte homogenates and in particulate subcellular fractions, but the percentage of cellular LPL activity that was released during incubation of myocytes with heparin was normal. In contrast, neutral lipase activity was increased in diabetic myocyte homogenates and microsomal fractions. Acid triacylglycerol lipase activity was not changed in diabetic myocytes. The decrease in LPL in myocytes owing to diabetes may result in the decreased functional LPL activity at the capillary endothelium of the diabetic heart.     

Intercellular transport of lysosomal acid lipase mediates lipoprotein cholesteryl ester metabolism in a human vascular endothelial cell-fibroblast coculture system.

We present results from studies of human cell culture models to support the premise that the extracellular transport of lysosomal acid lipase has a function in lipoprotein cholesteryl ester metabolism in vascular tissue. Vascular endothelial cells secreted a higher fraction of cellular acid lipase than did smooth muscle cells and fibroblasts. Acid lipase and lysosomal beta-hexosaminidase were secreted at approximately the same rate from the apical and basolateral surface of an endothelial cell monolayer. Stimulation of secretion with NH4Cl did not affect the polarity. We tested for the ability of secreted endothelial lipase to interact with connective tissue cells and influence lipoprotein cholesterol metabolism in a coculture system in which endothelial cells on a micropore filter were suspended above a monolayer of acid lipase-deficient (Wolman disease) fibroblasts. After 5-7 d, acid lipase activity in the fibroblasts reached 10%-20% of the level in normal cells; cholesteryl esters that had accumulated from growth in serum were cleared. Addition of mannose 6-phosphate to the coculture medium blocked acid lipase uptake and cholesterol clearance, indicating that lipase released from endothelial cells was packaged into fibroblast lysosomes by a phosphomannosyl receptor-mediated pathway. Supplementation of the coculture medium with serum was not required for lipase uptake and cholesteryl ester hydrolysis by the fibroblasts, but was necessary for cholesterol clearance. Results from our coculture model suggest that acid lipase may be transported from intact endothelium to cells in the lumen or the wall of a blood vessel. We postulate that delivery of acid hydrolases and lipoproteins to a common endocytic compartment may occur and have an impact on cellular lipoprotein processing.     

Lipoprotein lipase. Mechanism of formation of triglyceride-rich remnant particles from very low density lipoproteins and chylomicrons.

The catalytic rate of membrane-supported lipoprotein lipase has been determined for chylomicron and very low density lipoprotein substrates during the formation of triglyceride-depleted ("remnant") particles. Both lipoprotein species and their generated remnant products were competitive substrates for lipase activity. Remnant formation from each species was associated with decreasing kc but an unchanged apparent Km. This finding was confirmed from the rate of plot of total triglyceride catabolism by lipase at low substrate concentrations. When compared with the major very low density lipoprotein fraction (Sf 100-400), a fraction isolated from plasma with a lower flotation rate (Sf 40-100) had a lipid composition and decreased kc compatible with this representing a physiological remnant particle.     

Immunological studies on bovine milk lipoprotein lipase. Effects of Fab fragments on enzyme activity

Rabbit antiserum was prepared against purified bovine mild lipoprotein lipase. Immunoelectrophoresis of lipoprotein lipase gave a single precipitin line against the antibody which was coincident with enzyme activity. The gamma-globulin fraction inhibited heparin-releasable lipoprotein lipase activity of bovine arterial intima, heart muscle and adipose tissue. The antibody also inhibited the lipoprotein lipase activity from adipose tissue of human and pig, but not that of rat and dog. Fab fragments were prepared by papain digestion of the gamma-globulin fraction. Fab fragments inhibited the lipoprotein lipase-catalyzed hydrolysis of dimyristoylphosphatidylcholine vesicles and trioleoylglycerol emulsions to the same extent. The Fab fragments also inhibited the lipolysis of human plasma very low density lipoproteins. The change of the kinetic parameters for the lipoprotein lipase-catalyzed hydrolysis of trioleoylglycerol by the Fab fragments was accompanied with a 3-fold increase in Km and a 10-fold decrease in Vmax. Preincubation of lipoprotein lipase with apolipoprotein C-II, the activator protein for lipoprotein lipase, did not prevent inhibition of enzyme activity by the Fab fragments. However, preincubation with dipalmitoylphosphatidylcholine-emulsified trioleoylglycerol or Triton X-100-emulsified trioleoylglycerol had a protective effect (remaining activity 7.0 or 25.8%, respectively, compared to 1.0 or 0.4% with no preincubation). The addition of both apolipoprotein C-II and substrate prior to the incubation with the Fab fragments was associated with an increased protective effect against inhibition of enzyme activity; remaining activity with dipalmitoylphosphatidylcholine-emulsified trioleoylglycerol was 40.6% and with Triton X-100-emulsified trioleoylglycerol, 45.4%. Human plasma very low density lipoproteins also protected against the inhibition of enzyme activity by the Fab fragments. These immunological studies suggest that the interaction of lipoprotein lipase with apolipoprotein C-II in the presence of lipids is associated with a conformational change in the structure of the enzyme such that the Fab fragments are less inhibitory. The consequence of a conformational change in lipoprotein lipase may be to facilitate the formation of an enzyme-triacylglycerol complex so as to enhance the rate of the lipoprotein lipase-catalyzed turnover of substrate to products.     

Lipoprotein metabolism in hepatic lipase deficiency: studies on the turnover of apolipoprotein B and on the effect of hepatic lipase on high density lipoprotein

Hepatic lipase deficiency produces significant distortion in the plasma lipoprotein profile. Particles with reduced electrophoretic mobility appear in very low density lipoprotein (VLDL). Intermediate density lipoprotein (IDL) increases markedly in the circulation and plasma low density lipoprotein (LDL) levels fall. At the same time there is a mass redistribution within the high density lipoprotein (HDL) spectrum leading to dominance in the less dense HDL2 subfraction. The present study examines apolipoprotein B turnover in a patient with hepatic lipase deficiency. The metabolism of large and small very low density lipoproteins was determined in four control subjects and compared to the pattern seen in the patient. Absence of the enzyme did not affect the rate at which large very low density lipoproteins were converted to smaller particles within this density interval (i.e., of VLDL). However, subsequent transfer of small very low density lipoproteins to intermediate density particles was retarded by 50%, explaining the abnormal accumulation of VLDL in the patient's plasma. Despite this, intermediate density particles accumulated to a level 2.4-times normal because their subsequent conversion to low density lipoprotein has been almost totally inhibited. Consequently, the plasma concentration of low density lipoprotein was only 10% of normal. On the basis of these observations, hepatic lipase appears to be essential for the conversion of small very low density and intermediate density particles to low density lipoproteins. The pathways of direct plasma catabolism of these species were not affected by the enzyme defect. In vitro studies were performed by adding purified hepatic lipase to the patient's plasma.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activity of lipoprotein lipase in thyroidectomized rats

Total plasma postheparin lipolytic activity as well as lipoprotein lipase activity in plasma was higher after heparin injection in thyroidectomized rats than in controls. In contrast, the activity of liver lipase was lower in thyroidectomized rats. Adipose tissue from thyroidectomized rats contained more lipoprotein lipase activity than adipose tissue from controls as measured both in extracts of tissue homogenates and medium from in vitro incubations of tissue pieces. There were no differences between control and hypothyroid rats in the disappearance of intravenously injected 125I-labeled lipoprotein lipase, but when a low dose of heparin was injected before the labeled enzyme, the disappearance of 125I-labeled lipoprotein lipase was more retarded in thyroidectomized rats. The elimination of heparin itself was slightly retarded by thyroidectomy.     

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

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