Lipoprotein Lipase Concentration Research Articles

Activity and concentration of lipoprotein lipase in post-heparin plasma and the extent of coronary artery disease

Numerous studies have found polymorphisms in the lipoprotein lipase (LPL) gene to be associated with the risk of coronary artery disease (CAD), implicating LPL in the development of atherothrombotic disease. It remains controversial, however, whether LPL acts in a pro- or anti-atherogenic fashion. We quantitated activity and concentration of LPL in post-heparin plasma from 194 male patients undergoing coronary angiography. HDL cholesterol was significantly associated with LPL activity quartiles (1.09±0.26 the highest vs. 0.96±0.25 mmol/l the lowest quartile, P<0.01). There was also a trend towards higher total (5.61±1.33 vs. 5.16±1.44 mmol/l, P=0.059) and LDL cholesterol (3.92±1.39 vs. 3.46±1.06 mmol/l, P=0.09) with higher LPL activity. In contrast, measures of CAD extent showed no differences between LPL quartiles ( P>0.30 for prior myocardial infarction, number of diseased vessels, Gensini and extent scores). Additionally, there was no difference in LPL activity (CAD: n=158, 168±70 nmol/ml/min, no CAD: n=36, 180±89 nmol/ml/min, P=0.47) or concentration (280±121 ng/ml and 288±111 ng/ml, P=0.72) between patients with and without CAD. Our data show that, in spite of an association with lipoprotein parameters, LPL in post-heparin plasma is unrelated to the presence or the extent of CAD. Therefore, lipoprotein lipase determination in plasma does not appear to be a useful marker in the assessment of CAD risk.

Interleukin 6 and Haemostasis

Interleukin 6 and Haemostasis

Binding of Low Density Lipoproteins to Lipoprotein Lipase Is Dependent on Lipids but Not on Apolipoprotein B

Lipoprotein lipase (LPL) efficiently mediates the binding of lipoprotein particles to lipoprotein receptors and to proteoglycans at cell surfaces and in the extracellular matrix. It has been proposed that LPL increases the retention of atherogenic lipoproteins in the vessel wall and mediates the uptake of lipoproteins in cells, thereby promoting lipid accumulation and plaque formation. We investigated the interaction between LPL and low density lipoproteins (LDLs) with special reference to the protein-protein interaction between LPL and apolipoprotein B (apoB). Chemical modification of lysines and arginines in apoB or mutation of its main proteoglycan binding site did not abolish the interaction of LDL with LPL as shown by surface plasmon resonance (SPR) and by experiments with THP-I macrophages. Recombinant LDL with either apoB100 or apoB48 bound with similar affinity. In contrast, partial delipidation of LDL markedly decreased binding to LPL. In cell culture experiments, phosphatidylcholine-containing liposomes competed efficiently with LDL for binding to LPL. Each LDL particle bound several (up to 15) LPL dimers as determined by SPR and by experiments with THP-I macrophages. A recombinant NH(2)-terminal fragment of apoB (apoB17) bound with low affinity to LPL as shown by SPR, but this interaction was completely abolished by partial delipidation of apoB17. We conclude that the LPL-apoB interaction is not significant in bridging LDL to cell surfaces and matrix components; the main interaction is between LPL and the LDL lipids.

Fatty acid alcohol ester-synthesizing activity of lipoprotein lipase.

The fatty acid alcohol ester-synthesizing activity of lipoprotein lipase (LPL) was characterized using bovine milk LPL. Synthesizing activities were determined in an aqueous medium using oleic acid or trioleylglycerol as the acyl donor and equimolar amounts of long-chain alcohols as the acyl acceptor. When oleic acid and hexadecanol emulsified with gum arabic were incubated with LPL, palmityl oleate was synthesized, in a time- and dose-dependent manner. Apo-very low density lipoprotein (apoVLDL) stimulated LPL-catalyzed palmityl oleate synthesis. The apparent equilibrium ratio of fatty acid alcohol ester/oleic acid was estimated using a high concentration of LPL and a long (20 h) incubation period. The equilibrium ratio was affected by the incubation pH and the alcohol chain length. When the incubation pH was below pH 7.0 and long chain fatty acyl alcohols were used as substrates, the fatty acid alcohol ester/free fatty acid equilibrium ratio favored ester formation, with an apparent equilibrium ratio of fatty acid alcohol ester/fatty acid of about 0.9/0.1. The equilibrium ratio decreased sharply at alkaline pH (above pH 8.0). The ratio also decreased when fatty alcohols with acyl chains shorter than dodecanol were used. When a trioleoylglycerol/fatty acyl alcohol emulsion was incubated with LPL, fatty acid alcohol esters were synthesized in a dose- and time-dependent fashion. Fatty acid alcohol esters were easily synthesized from trioleoylglycerol when fatty alcohols with acyl chains longer than dodecanol were used, but synthesis was decreased with fatty alcohols with acyl chain lengths shorter than decanol, and little synthesizing activity was detected with shorter-chain fatty alcohols such as butanol or ethanol.

Lipoprotein lipase transport in plasma: role of muscle and adipose tissues in regulation of plasma lipoprotein lipase concentrations

Lipoprotein lipase (LPL) is synthesized in tissues involved in fatty acid metabolism such as muscle and adipose tissue. LPL is also found in the circulation, but is mostly lipolytically inactive. The proportion of active circulating LPL increases after a fatty meal. We investigated the release of active and inactive LPL from adipose tissue and muscle in the fasting and postprandial states. Arteriovenous concentration gradients of LPL across adipose tissue and forearm muscle were measured in male subjects before and after a fat-rich meal (n = 7) and before and during infusion of a triacylglycerol emulsion (Intralipid) (n = 6). Plasma LPL activity rose after the meal and more so during Intralipid infusion. Plasma LPL mass (>95% inactive LPL) increased after the meal but decreased after Intralipid infusion. In the fasting state (n = 13) muscle efflux of LPL activity was 0.263 +/- 0.098 mU/min per 100 ml of muscle tissue whereas there was an influx of LPL activity to adipose tissue of 0.085 +/- 0.100 mU/min per 100 g of adipose tissue (P < 0. 02 muscle vs. adipose tissue). Similarly in the postprandial state only muscle released LPL activity. Both tissues released LPL mass. In the fasting state efflux was 17.8 +/- 8.8 ng/min per 100 ml muscle and 55.2 +/- 21.3 ng/min per 100 g of adipose tissue (P < 0. 05 muscle vs. adipose tissue). Release of LPL, either active or inactive, was not correlated with levels of non-esterified fatty acids or plasma triacylglycerol. In conclusion, there is a substantial release of LPL from adipose tissue and muscle, most of which is inactive. A small proportion of active LPL seems to be redistributed from muscle to adipose tissue.

Serum lipoprotein lipase in healthy subjects: effects of gender and age, and relationships to lipid parameters.

Using serum samples obtained from normal individuals who had not received heparin injection, we investigated the relationship of lipoprotein lipase (LPL) to lipid and apolipoprotein concentrations in serum. For the measurement of LPL concentration, a highly sensitive enzyme-linked immunosorbent assay (ELISA) was developed, in which the lowest detection limit was 9 micrograms/L. The mean (SD) serum LPL concentration was 50.7 (14.9) micrograms/L (n = 240). It was lower in men [45.5 (14.0) micrograms/L in men and 56.0 (13.9) micrograms/L in women]. Serum LPL concentration correlated positively with high-density lipoprotein cholesterol (HDL-C, r = 0.549) and apolipoprotein AI (apoAI, r = 0.487), and inversely with triglycerides (r = -0.423). Our results suggest that serum LPL may be a factor in the modulation of HDL-C and triglyceride concentrations in normal subjects.

Interaction of lipoprotein lipase with homogeneous lipid emulsions

The central function of lipoprotein lipase (LpL) is to hydrolyze triacylglycerols in chylomicrons and very low density lipoproteins. We have examined the binding of purified milk lipoprotein lipase to homogeneous synthetic lipid emulsions. Emulsions composed of either naturally occurring ester-linked lipids or the non-hydrolyzable ether analogues were prepared by sonication and pressure extrusion, and fractionated by sucrose density gradient centrifugation. Flotation analysis using the analytical ultracentrifuge indicated that the individual fractions were relatively homogeneous with respect to size with flotation coefficients and molecular weights for the separated fractions ranging from 100 to 1100 S and 5.2 x 10(7) to 6.0 x 10(8), respectively. Purified milk lipoprotein lipase bound with high affinity and in a saturable manner to emulsions prepared from the non-hydrolyzable ether-linked lipid analogues of 1-oleoyl, 2-palmitoyl phosphatidylcholine and triolein. At low concentrations of LpL, the enzyme caused aggregation of the emulsion particles by interparticle cross-linking. At higher LpL concentrations, the flotation coefficient of the emulsions decreased significantly with a concomitant increase in particle density. At saturation, the number of LpL monomers bound to lipid particles of radii 67, 75, and 79 nm was 1315, 1449, and 1466, respectively. The results demonstrate close packing of LpL on the lipid surface and are consistent with there being little disruption to the overall structure of the emulsion particle.

Uptake of hypertriglyceridemic very low density lipoproteins and their remnants by HepG2 cells: the role of lipoprotein lipase, hepatic triglyceride lipase, and cell surface proteoglycans

Hypertriglyceridemic very low density lipoproteins (HTG-VLDL, S(f) 60-400) are not taken up by HepG2 cells. However, addition of bovine milk lipoprotein lipase (LPL) at physiological concentrations markedly stimulates uptake. In the present study, we determined whether: a) LPL catalytic activity is required for uptake, b) LPL functions as a ligand, and c) cell surface hepatic triglyceride lipase (HL) and/or proteoglycans are involved. Incubation of HepG2 cells with HTG-VLDL plus LPL (8 ng/ml) increased cellular cholesteryl ester (CE) 3.5-fold and triglyceride (TG) 6-fold. Heat-inactivation of LPL abolished the effect. Addition of tetrahydrolipstatin (THL, an LPL active-site inhibitor) to HTG-VLDL + LPL, inhibited the cellular increase in both CE and TG by greater than 90%. Co-incubation of HTG-VLDL + LPL with heparin, heparinase, or heparitinase, blocked CE accumulation by 70%, 48%, and 95%, respectively, but had no effect on the increase in cellular TG. Pre-treatment of cells with 1 mM 4-methylumbelliferyl-beta-D-xyloside, (beta-xyloside) to reduce cell surface proteoglycans inhibited the increase in CE induced by HTG-VLDL + LPL by 78%. HTG-VLDL remnants, prepared in vitro and isolated free of LPL activity, stimulated HepG2 cell CE 2.8-fold in the absence of added LPL, a process inhibited with THL by 66%. Addition of LPL (8 ng/ml) to remnants did not further enhance CE accumulation. HepG2 cell HL activity, released by heparin, was inhibited 95% by THL. The amount of HL activity and immunoreactive mass, released by heparin, was reduced 50-60% in beta-xyloside-treated cells. These results indicate that physiological concentrations of LPL promote HepG2 cell uptake of HTG-VLDL primarily due to remnant formation and that LPL does not play a major role as a ligand. HL activity and cell surface proteoglycans significantly enhance the subsequent uptake of VLDL remnants.

Regulatory effects of lipoprotein lipase on proliferative and cytotoxic activity of NK cells

Lipoprotein lipase (LPL) induced, in a dose-dependent fashion, a 2-fold and 11-fold increase in the proliferative response of peripheral blood lymphocytes (PBL) at 48 and 72 h, respectively; a 4- and 12-fold increase in natural killer (NK) cells, respectively; and a maximal 3-fold induction in interleukin-2 (IL-2)-treated NK cells at 72 h. T lymphocytes did not proliferate independently of the concentration of LPL used. LPL decreased the proliferative response of K562 and U937 cell lines. The effect on NK cells could be blocked by anti-LPL if it was added before LPL binding to the cell membrane. Contrary to its effects on NK proliferative response, LPL inhibited spontaneous cytotoxicity and lymphokine-activated killer activity (LAK). The effect was dose-dependent, target-dependent (U937 was more sensitive than K562 in LAK assays), but not LPL-binding time-dependent. Treatment of NK cells with heparinase overcame the inhibitory effect of LPL in spontaneous cytotoxicity. LPL binding to cell membranes, as assessed by flow cytometry, was as follows: K562 cells > monocytes > NK cells > LAK cells > U937 cells, absent in T lymphocytes and partially sensible to heparinase and IL-2 treatments. Protein kinase C translocation was observed upon treatment of NK cells with LPL. Three proteins in NK cell membrane (76, 57.2, and 27.2 kD), two in the cytosol (57.2 and 27.2 kD), and only one in ANA-1 cell membrane (76 kD) were precipitated with LPL-Sepharose. LPL receptors seem to be responsible for the proliferative and cytotoxic response observed in LPL-stimulated NK cells.

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.

Trafficking of Dietary Fat in Lean Rats

Despite increasing interest in the role that fuel partitioning plays in determining body composition, the relative importance of oxidative versus storage pathways in the clearance of dietary fat remains unclear. A widely held view is that the primary destination of chylomicron triglyceride fatty acids (TGFA) is adipose tissue, and the primary source of lipid fuel for skeletal muscle is non-esterified fatty acids (NEFA). An alternate view is that muscle, not adipose tissue, is the primary site of TGFA clearance. This view is supported by estimates of the total lipoprotein lipase content of muscle and adipose tissue. To directly study the partitioning of dietary fat between oxidation and storage, 14C-labeled oleic acid was fed to Sprague Dawley rats and its metabolic rate followed over 30 days. Two hours after ingestion, more than 3.5 times as much label was found in skeletal muscle tissue (2.42 +/- 0.45 nmols) and CO2 (0.25 +/- 0.01 nmols) than was found in adipose tissue (0.71 +/- 0.14 nmols). Intramuscular triglyceride was the lipid class most extensively labeled. After skeletal muscle, liver was the next most important site of TGFA clearance. Surprisingly a substantial quantity of label remained associated with the GI tract even 24 hours after ingestion. Between 2 and 10 days following ingestion there was a net decline in the 14C content of muscle, liver and GI tract, associated with a net rise in the 14C content of adipose tissue. These findings demonstrate: 1) the importance of skeletal muscle and liver in whole organism TGFA clearance, 2) the importance of intramuscular partitioning of lipid fuels between direct oxidation and storage as TG, 3) the potentially important role of the GI tract in the delivery of dietary fat to the circulation 10-24 hours following ingestion, and 4) the stability of adipose tissue as a storage site. The complex nature of the tissue-specific clearance of TGFA over time is perhaps better described by the term "trafficking" than by the more commonly used term "partitioning." Future studies of TGFA clearance combined with sampling of relevant tissues over time will provide insight into the specific roles that abnormalities in liver, muscle and adipose tissue TGFA metabolism play in the development of hypertriglyceridemic disorders and states of increased or reduced body weight.

ヒト肝性トリグリセリドリパーゼ(HTGL)の生理作用の解明

The purpose of this study is to evaluate changes in hepatic triglyceride lipase (HTGL) and lipoprotein lipase (LPL) as a cause of dyslipoproteinemia in hypothyroidism. Hypothyroidism is associated with pronounced hypercholesterolemia and moderate hypertriglyceridemia. Recent studies suggest that such changes in serum lipids result from the decrease in LDL-receptors and also HTGL and/or LPL activities. In order to accurately evaluate the correlation between both lipases and lipoprotein abnormalities in patients with hypothyroidism, we measured LPL and HTGL mass concentrations in postheparin plasma by means of a sandwich-enzyme immunoassay (EIA) using two distinct monoclonal antibodies before and after treatment with thyroid hormone.Seven patients with untreated hypothyroidism and euthyroid normal controls (n=76) were studied. Hypothyroidism group (H) consisted of 3 males and 4 females (age 60±5 years) and the control group (N) of 46 males and 30 females (age 42±13 years). The plasma cholesterol (C) and triglyceride (TG) levels were significantly increased in the H group compared to the N group: 327±27 vs. 192±27ma/dl and 145±24 vs 88±33mg/dl, respectively. There was no difference in HDL-C (61.2±5.7 vs. 63.1±6.6mg/dl). HTGL was markedly reduced (79%) in the H group (264±57 vs. 1, 252±55ng/ml), while LPL was only slightly reduced (16%) (153±17 vs. 182±5ng/ml). The H group showed a hyperlipidemia of IIa (n=5) or IIb (n=2) as a WHO phenotype. After the patients were treated with thyroid hormone, HTGL level was drastically increased from 264±57ng/ml to 858±115ng/ml, and the LPL level also increased from 153±17ng/ml to 201±29ng/ml. Plasma C level decreased from 327±27 to 234±3mg/dl, and TG from 145±24 to 78±13mg/dl. By plotting all the patient data before and after the treatment, HTGL showed a positive correlation with FT3 (r=0.86) and negative correlation with TSH (r=-0.66). HTGL showed significant inverse correlations with LDL-TG/apoB (r=-0.74) and HDL-TG/apoA (r=-0.74). Our data indicates that HTGL mass concentration is regulated by thyroid hormone level leading us to conclude that HTGL is closely related to the metabolism of triglyceride-rich LDL and HDL particles.

Lipoprotein Lipase Facilitates Very Low Density Lipoprotein Binding to the Subendothelial Cell Matrix

The effect of bovine lipoprotein lipase (LPL) on very low density lipoprotein (VLDL) binding to subendothelial matrix was studied. Without LPL, VLDL bound poorly to the matrix. However, decreasing NaCl or elevating Ca++ concentration increased matrix VLDL binding. With LPL, VLDL binding was markedly increased. Since LPL is a normal constituent of the artery wall and is elevated in atherosclerotic lesions, we postulate two potential mechanisms for the involvement of VLDL and LPL in atherogenesis. First, VLDL acquisition is attenuated by the increased matrix LPL content in the developing atheroma. Secondly, elevated plasma levels of VLDL (and VLDL remnants) such as in Type II or III dyslipidemia could enhance such interactions. These events likely accelerate the rate of atherosclerosis lesion development.

In vivo removal of beta-VLDL, chylomicron remnants, and alpha 2-macroglobulin in the rat.

The low density lipoprotein receptor-related protein (LRP)/alpha 2-macroglobulin (alpha 2M) receptor has been suggested as a potential chylomicron remnant receptor. To investigate the involvement of LRP in chylomicron remnant metabolism in vivo, cross-competition experiments with chylomicron remnants, beta-VLDL, and receptor-active alpha 2M, complexed with trypsin (alpha 2M-trypsin), were performed in rats. Saturating concentrations of unlabeled beta-VLDL failed to inhibit the plasma clearance and hepatic uptake of 125I-labeled alpha 2M-trypsin and, vice versa, alpha 2M-trypsin failed to retard the removal of 125I-labeled chylomicron remnants. It has been demonstrated previously that bovine lipoprotein lipase (LPL) strongly enhances the binding of apolipoprotein E-containing lipoproteins to LRP (U. Beisiegel, W. Weber, and G. Bengtsson-Olivecrona. 1991. Proc. Natl. Acad. Sci. USA. 88: 8342-8346). Therefore, beta-VLDL were enriched with isolated LPL or heparin was injected simultaneously with beta-VLDL to increase the concentration of endogenous LPL bound to beta-VLDL. Yet, no inhibition of the plasma elimination and the hepatic uptake of 125I-labeled alpha 2M-trypsin was observed after injection of saturating amounts of beta-VLDL enriched with LPL. We conclude that in the rat triglyceride-rich lipoproteins and alpha 2M-trypsin bind in vivo either to different binding domains of LRP or to a different receptor protein.

Changes in particle size of high density lipoproteins during incubation with very low density lipoproteins, cholesteryl ester transfer protein and lipoprotein lipase

Previous reports have produced conflicting views of the effects of lipoprotein lipase (LPL) on the particle size distribution of high density lipoproteins (HDL). In this study we have investigated the changes in particle size of HDL promoted by the interaction of LPL, the cholesteryl ester transfer protein (CETP) and very low density lipoproteins (VLDL). When the plasma fraction of d < 1.21 g/ ml (containing all lipoprotein fractions) was incubated for 24 h with bovine milk LPL alone or with CETP alone, there was relatively little change in the particle size distribution of HDL. When both LPL and CETP were added to the lipoprotein mixture, there was a substantial reduction in the particle size of HDL. This reduction in HDL particle size was found to be a direct function of the concentration of CETP. It was also influenced by the concentrations of VLDL and LPL, although in these cases the relationships were complex. When mixtures of the plasma fraction of d = 1.006−1.21 g/ ml (this fraction includes low density lipoproteins and HDL but not VLDL) were supplemented with both LPL and CETP and incubated in the presence of varying concentrations of added VLDL, there was a progressive increase in the conversion of HDL into very small HDL particles of radius 3.7 nm as the concentration of VLDL triacylglycerol increased up to about 400 nmol/nml. However, further increases in the concentration of VLDL were accompanied by a progressive reduction in the formation of small HDL particles until, at higher VLDL concentrations, the effect was all but abolished. There was a similar enhancement in the formation of small HDL when LPL was added at low but not at high concentrations. These findings are consistent with the existence of two opposing processes. On the one hand there is likely to be a synergism between CETP and the non-esterified fatty acids (NEFA) released by LPL; this will favour a reduction in HDL particle size. On the other hand, the transfer of lipolysis products from VLDL to HDL may mask any such particle size reduction. The fact that the reduction in HDL particle size promoted by LPL, CETP and VLDL was found to be all but abolished by adding fatty acid-poor albumin to the incubation mixture is consistent with the proposition that NEFA are involved in the process. It also suggests, however, that the phenomenon may have little if any physiological significance.

Transport of free fatty acid and triglyceride in anhepatic rats

Without a liver the fractional plasma removal of free fatty acid is one third and chylomicron triglyceride one half of that in the intact rat. The intestine of the anhepatic rat converted enteral fatty acid to plasma triglyceride but was unable to do the same for plasma free fatty acid. This decrease in plasma free fatty acid removal and the inability to recycle the acid as triglyceride were, in part, responsible for the conversion of large fractions of plasma triglyceride flux to the plasma free acid in hepatectomized rats. Increased intravascular triglyceride lipolysis resulting from high circulating lipoprotein lipase concentrations and reduced plasma triglyceride removal were other factors shifting the partition of anhepatic plasma fatty-acid transport from the ester to the free. After the anhepatic plasma clearance of either free fatty acid or triglyceride, relatively more of both compounds was recovered in the lipid of actively metabolizing (heart and muscle) as opposed to storage (adipose) tissue when compared with controls. Sequential evaluations of the recovery of plasma free fatty acid and triglyceride in tissues of anhepatic rats demonstrated accumulation or storage solely in adipose tissue and only when the plasma fatty acid was in triglyceride. This observation and the large conversion of anhepatic circulating triglyceride to the free acid may, in part, explain the lack of an increase in adipose lipid with reduced hepatic mass. The data help explain the preferential use of a lipid fuel in liver disease and the difficulties in obtaining carbon storage in this condition.

Lipoprotein lipase stored in adipocytes and muscle cells is a cryptic enzyme.

The status of lipoprotein lipase (LPL) has been examined in different cell types (adipose, skeletal muscle, and heart muscle cells) and different tissues (adipose, muscle, and cardiac tissues) from mouse, rat, and human. Cell and secreted activities were compared in cycloheximide-, heparin-treated cells present in culture. A gross underestimation of cell LPL activity was found; excess of LPL over substrate and/or apolipoprotein C-II was excluded as well as inhibition by cell component(s) or detergent molecules used to disrupt membrane structures in the cell lysates. Unmasking of LPL activity occurred upon dilution: the higher the concentration of LPL, the higher were the dilution factor and the concentration of heparin required to reach a plateau of activity. This maximal value was found to be identical to that determined in the secretion medium, indicating that the cell LPL activity can be determined in toto. The unmasking effect of dilution upon LPL activity was extended to adipose, muscle, and cardiac tissues from rat and to adipose tissues from mouse and human. In agreement with previous results (Vannier et al., 1989, J. Biol. 264: 13199-13205), our results are in favor of LPL as being cryptic within the cell. A model is proposed, in which potentially active LPL molecules are present as aggregates in various membrane compartments. It is concluded that the determination of the pool size of catalytically active cell LPL has to be estimated in vitro under the appropriate conditions described herein.

A sensitive, inexpensive method for determining minute quantities of lipase activity

We describe a sensitive and reproducible lipase assay based on the binding of 63Ni to fatty acid. This method can detect down to 1 nmol of fatty acid per milliliter of solution. It has been adapted for measuring low concentrations of lipoprotein lipase and hepatic triacylglycerol lipase. Furthermore, in the presence of tritiated triolein, the method is insensitive to radiolabel interference, even when the fatty acid is labeled.

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 μg/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.

Metabolic fate of rat heart endothelial lipoprotein lipase.

When isolated rat hearts were perfused with medium containing 125I-labeled bovine lipoprotein lipase (LPL), they bound both lipase activity and radioactivity. More than 80% of the bound lipase could be rapidly released by heparin. Low concentrations of bovine LPL displaced 50-60% of the endogeneous, endothelial-bound LPL. Higher concentrations caused additional binding. Both binding and exchange were rapid processes. The hearts continuously released endogenous LPL into the medium. An antiserum that inhibited bovine but not rat LPL was used to differentiate endogeneous and exogeneous LPL activity. When the pool of endothelial LPL was labeled with bovine 125I-labeled LPL and then chased with unlabeled bovine LPL, approximately 50% of the labeled lipase was rapidly displaced. During chase perfusion with medium only, catalytically active bovine LPL appeared in the perfusate. The rate of release was similar to that observed for endogeneous LPL activity and amounted to 10-13% of the heparin-releasable fraction in the first 5 min of perfusion. There was little or no degradation of bovine 125I-labeled LPL to fragments or acid-soluble products. These results indicate that endothelial LPL is accessible for exchange with exogeneous LPL and that detachment rather than degradation may be the pathway for catabolism of endothelial LPL.