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Biomedical subjects

G Olivecrona

Publications and source records attributed to G Olivecrona.

At least 19 recordsLinked to original sources
PubMed2002

Peroxisome proliferator-activated receptor (PPAR) agonists decrease lipoprotein lipase secretion and glycated LDL uptake by human macrophages.

Lipoprotein lipase (LPL) acts independently of its function as triglyceride hydrolase by stimulating macrophage binding and uptake of native, oxidized and glycated LDL. Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors expressed in monocyte/macrophages, where they control cholesterol homeostasis. Here we study the role of PPARs in the regulation of LPL expression and activity in human monocytes and macrophages. Incubation of human monocytes or macrophages with PPARalpha or PPARgamma ligands increases LPL mRNA and intracellular protein levels. By contrast, PPAR activators decrease secreted LPL mass and enzyme activity in differentiated macrophages. These actions of PPAR activators are associated with a reduced uptake of glycated LDL and could influence atherosclerosis development associated with diabetes.

Biological Transport↗
PubMed2001

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.

Animals↗
PubMed2001

Effects of the heparin-mimicking compound RG-13577 on lipoprotein lipase and on lipase mediated binding of LDL to cells.

Lipoprotein lipase (LPL) has high affinity for heparin and heparin-like compounds. In vivo the enzyme is attached to heparan sulfate proteoglycans on the endothelium of capillaries and larger blood vessels. The enzyme is released from these sites after intravenous injection of heparin. One has here investigated the effects of RG-13577 on LPL, both after intravenous injection to rats and under cell culture conditions. RG-13577 is a heparin-mimicking compound known to prevent angiogenesis by interference with binding of growth factors to cells. It has therefore been considered for use in cancer therapy as well as for prevention of atherosclerosis and restenosis. It was found that intravenously injected RG-13577 released both LPL and hepatic lipase (HL) to the blood. Binding of LPL in extrahepatic tissues was prevented and clearance of radiolabeled LPL from the circulation was delayed. Furthermore, RG-13577 released LPL from extracellular matrix (ECM) produced by endothelial cells and from THP-1 monocyte-derived macrophages. Lipase-mediated binding and uptake of human LDL in these cells was also prevented by RG-13577. Thus, in the test systems RG-13577 had the same effects as heparin, but on a molar basis RG-13577 was in all cases less effective.

Animals↗
PubMed2001

Protease inhibitors, the responsible components for the serum-dependent enhancement of Actinobacillus actinomycetemcomitans leukotoxicity.

Serum enhances the leukotoxic activity of Actinobacillus actinomycetemcomitans against human polymorphonuclear leukocytes (PMNL) by a mechanism that still is unknown. Early attempts to identify the serum components responsible for this enhancement gave no conclusive results, but indicated that the lipoprotein-containing fraction of the serum was involved in the interaction. This study aimed to clarify the role of serum lipoproteins in the leukotoxin interaction, and to identify other serum components involved. The main hypothesis examined was that the leukotoxicity enhancement might depend on serum protease inhibitors that block proteolytic cleavage of leukotoxin by enzymes released from the leukocytes. PMNL were isolated from human peripheral blood and incubated with purified leukotoxin in the presence of serum or purified serum components or lipoprotein-deficient serum. Leukotoxin was also incubated with purified elastase and cathepsin G or with enzyme mixtures from degranulated PMNL. The leukotoxic activity in these mixtures was determined as the extracellular release of lactate dehydrogenase from PMNL. Cleavage of the toxin was showed by gel electrophoresis and Western blot. Morphological changes in PMNL from the above mixtures were examined by electron microscopy. Enzymes from degranulated PMNL cleaved leukotoxin to non-cytotoxic fragments. Elastase and cathepsin G were mainly responsible for the cleavage. Inhibition of leukotoxin degradation was found in the presence of whole serum or of the serum protease inhibitors alpha2-macroglobulin and alpha1-proteinase inhibitor. Under these conditions enhanced PMNL lysis was also observed. A similar enhancement of PMNL lysis was found when PMNL degranulation was blocked by EDTA. On the other hand, lipoprotein-deficient serum had no influence on the leukotoxic activity. The results indicate that the increased leukotoxicity of A. actinomycetemcomitans observed in the presence of human serum is caused by the serum protease inhibitors that counteract proteolytic degradation of leukotoxin. The degradation is caused by enzymes from degranulated PMNL triggered by leukotoxin.

Aggregatibacter actinomycetemcomitans↗
PubMed2001

Food deprivation increases post-heparin lipoprotein lipase activity in humans.

OBJECTIVE: To study the effect of fasting on lipoprotein lipase (LPL) activity in human post-heparin plasma, representing the functional pool of LPL. DESIGN: Fourteen healthy volunteers were recruited for the study. The subjects were fasted for 30 h. Activities of LPL and hepatic lipase (HL), and LPL mass, were measured in pre- and post-heparin plasma in the fed and in the fasted states, respectively. For comparison, LPL and HL activities were measured in pre- and post-heparin plasma from fed and 24-h-fasted guinea pigs. RESULTS: Fasting caused a significant drop in the levels of serum insulin, triglycerides and glucose in the human subjects. Post-heparin LPL activity increased from 79 +/- 6.4 mU mL-1 in the fed state to 112 +/- 10 mU mL-1 in the fasted state (P < 0.01), while LPL mass was 361 +/- 29 in the fed state and 383 +/- 28 in the fasted state, respectively (P = 0.6). In contrast, fasting of guinea pigs caused an 80% drop in post-heparin LPL activity. The effect of fasting on human and guinea pig post-heparin HL activity were moderate and statistically not significant. CONCLUSIONS: In animal models such as rats and guinea pigs, post-heparin LPL activity decreases on fasting, presumably due to down-regulation of adipose tissue LPL. In humans, fasting caused increased post-heparin LPL activity.

Adipose Tissue↗
PubMed2001

Lipoprotein lipase during continuous heparin infusion: tissue stores become partially depleted.

Lipoprotein lipase (LPL) and hepatic lipase (HL) are located at vascular surfaces in extrahepatic tissues and in the liver, respectively. Heparin displaces the enzymes into the circulating blood. Animal studies have shown that the liver takes up and degrades LPL. To explore whether heparin leads to a depletion of tissue stores, we followed the lipase activities in plasma during an 8-hour primed infusion of heparin in 10 healthy subjects. After an initial peak, the HL activity decreased slowly after a time curve similar to that for activated partial thromboplastin time. The time curve for LPL was different. After the initial peak, the activity dropped by almost 80%, from 30 to 120 minutes, and then leveled off to a plateau that corresponded to about 15% of the peak level. A second bolus of heparin was given to 4 subjects after 4 hours. The plasma LPL activity increased, but only to about 35% of the original peak level. We conclude that when heparin releases LPL into plasma, the lipase becomes liable to be taken up and degraded by the liver. After less than 1 hour, the stores of LPL have been exhausted, and recruitment of lipase into plasma depends on a slow but stable delivery of newly synthesized molecules.

Aged↗
PubMed2000

Inhibition of lipoprotein lipase by alkanesulfonyl fluorides.

A number of alkanesulfonyl halides (chlorides and fluorides) and esters were synthesized and their effect on the activity of lipoprotein lipase (LPL) was studied. Sulfonyl fluorides proved to be efficient inhibitors of LPL when the enzyme was incubated with a 10-fold molar excess of the inhibitors in a buffer containing bile salts (deoxycholate). Hexadecane- and dodecanesulfonyl fluorides caused 50% inhibition of LPL activity at concentrations of 10 to 20 microM.

Alkanesulfonates↗
PubMed2000

Apolipoprotein CII from rainbow trout (Oncorhynchus mykiss) is functionally active but structurally very different from mammalian apolipoprotein CII.

Apolipoprotein CII (apoCII) plays an important role in plasma lipid metabolism as an activator for lipoprotein lipase (LPL). We have amplified and sequenced apoCII cDNA from rainbow trout. Amino acid sequence analyses confirmed that this sequence corresponded to the protein that had apoCII activity. Northern blot analyses showed that apoCII mRNA was present in both liver and intestine, but the level in intestine was very low. Two major transcripts (800 and 600bp) were found. The predicted amino acid sequence consists of 112 amino acid residues, including the signal peptide. The mature peptide is seven residues longer than human apoCII (86 versus 79 residues) due to an extension at the amino-terminal end. The rainbow trout sequence showed an overall identity of only 20-25% to previously known apoCII sequences. The carboxy-terminal region (residues 51-79, human numbering) showed 35-45% identity to other apoCII sequences, while in the amino-terminal region, there was little if any identity and it was not possible to predict any long amphipathic, potentially lipid-binding alpha-helices. Trout apoCII was present in all lipoprotein fractions including LDL. At +10 degrees C trout plasma showed higher ability to stimulate LPL than human plasma. We conclude that apoCII from rainbow trout is in most parts structurally different from apoCII from other species, and that it is adapted to function at low temperature.

Amino Acid Sequence↗
PubMed2000

Characterization of recombinant wild type and site-directed mutations of apolipoprotein C-III: lipid binding, displacement of ApoE, and inhibition of lipoprotein lipase.

The physicochemical properties of recombinant wild type and three site-directed mutants of apolipoprotein C-III (apoC-III), designed by molecular modeling to alter specific amino acid residues implicated in lipid binding (L9T/T20L, F64A/W65A) or LPL inhibition (K21A), were compared. Relative lipid binding efficiencies to dimyristoylphosphatidylcholine (DMPC) were L9T/T20L > WT >K21A > F64A/W65A with an inverse correlation with size of the discoidal complexes formed. Physicochemical analysis (Trp fluorescence, circular dichroism, and GdnHCl denaturation) suggests that L9T/T20L forms tighter and more stable lipid complexes with phospholipids, while F64A/W65A associates less tightly. Lipid displacement properties were tested by gel-filtrating apoE:dipalmitoylphosphatidylcholine (DPPC) discoidal complexes mixed with the various apoC-III variants. All apoC-III proteins bound to the apoE:DPPC complexes; the amount of apoE displaced from the complex was dependent on the apoC-III lipid binding affinity. All apoC-III proteins inhibited LPL in the presence or absence of apoC-II, with F64A/W65A displaying the most inhibition, suggesting that apoC-III inhibition of LPL is independent of lipid binding and therefore of apoC-II displacement. Taken together. these data suggest that the hydrophobic residues F64 and W65 are crucial for the lipid binding properties of apoC-III and that redistribution of the N-terminal helix of apoC-III (L9T/T20L) enhances the stability of the lipid-bound protein, while LPL inhibition by apoC-III is likely to be due to protein:protein interactions.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2000

Contribution of the carboxy-terminal domain of lipoprotein lipase to interaction with heparin and lipoproteins.

The C-terminal domain of lipoprotein lipase (LPL) is involved in several important interactions. To assess its contribution to the binding ability of full-length LPL we have determined kinetic constants using biosensor technique. The affinity of the C-terminal domain for heparin was about 500-fold lower than that of full-length LPL (K(d) = 1.3 microM compared to 3.1 nM). Replacement of Lys403, Arg405 and Lys407 by Ala abolished the heparin affinity, whereas replacement of Arg420 and Lys422 had little effect. The C-terminal domain increased binding of chylomicrons and VLDL to immobilized heparin relatively well, but was less than 10% efficient in binding of LDL compared to full-length LPL. Deletion of residues 390-393 (WSDW) did not change the affinity to heparin and only slightly decreased the affinity to lipoproteins. We conclude that the C-terminal folding domain contributes only moderately to the heparin affinity of full-length LPL, whereas the domain appears important for tethering triglyceride-rich lipoproteins to heparin-bound LPL.

Animals↗
PubMed2000

Nutritional regulation of binding sites for lipoprotein lipase in rat heart.

Several laboratories have shown that when rats are fasted, the amount of lipoprotein lipase (LPL) at the vascular endothelium in heart (monitored as the amount released by heparin) increases severalfold without corresponding changes in the production of LPL. This suggests that there is a change in endothelial binding of LPL. To study this, (125)I-labeled bovine LPL was injected. The fraction that bound in the heart was more than twice as high in fasted than in fed rats, 4.3% compared with 1.9% of the injected dose. Refeeding reversed this in 5 h. When unlabeled LPL was injected before the tracer, the fraction of (125)I-LPL that bound in heart decreased, indicating that the binding was saturable. When isolated hearts were perfused at 4 degrees C with a single pass of labeled LPL, twice as much bound in hearts of fasted rats. We conclude that fasting causes a change in the vascular endothelium in heart such that its ability to bind LPL increases.

Adipose Tissue↗
PubMed2000

Functional characterization of 4 polymorphisms in promoter region of hepatic lipase gene.

Hepatic lipase (HL) is a lipolytic enzyme involved in the metabolism of plasma lipoproteins, especially high density lipoproteins. Association studies have provided strong evidence for relations of common mutations in the promoter region of the HL gene to postheparin plasma HL activity and the plasma high density lipoprotein cholesterol concentration, but the functional relevance of these polymorphisms has not been evaluated to date. We analyzed the physiological significance of 4 common polymorphisms (-250G/A, -514C/T, -710T/C, and -763A/G, all in strong linkage disequilibrium) in the promoter of the HL gene by use of electrophoretic mobility shift assays and transient transfection studies in HepG2 cells. No consistent evidence was found for a significant contribution of any of these polymorphisms to the basal rate of transcription of the HL gene. These data suggest that the 4 polymorphisms in the promoter region of the HL gene are in linkage disequilibrium with >/=1 as-yet-unknown functional polymorphisms in the HL gene locus with a significant effect on HL metabolism and/or enzymatic activity.

Adult↗
PubMed1999

The second and fourth cluster of class A cysteine-rich repeats of the low density lipoprotein receptor-related protein share ligand-binding properties.

The low density lipoprotein receptor-related protein (LRP) is a multifunctional endocytic cell-surface receptor that binds and internalizes a diverse array of ligands. The receptor contains four putative ligand-binding domains, generally referred to as clusters I, II, III, and IV. In this study, soluble recombinant receptor fragments, representing each of the four individual clusters, were used to map the binding sites of a set of structurally and functionally distinct ligands. Using surface plasmon resonance, we studied the binding of these fragments to methylamine-activated alpha(2)-macroglobulin, pro-urokinase-type plasminogen activator, tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor-1, t-PA.plasminogen activator inhibitor-1 complexes, lipoprotein lipase, apolipoprotein E, tissue factor pathway inhibitor, lactoferrin, the light chain of blood coagulation factor VIII, and the intracellular chaperone receptor-associated protein (RAP). No binding of the cluster I fragment to any of the tested ligands was observed. The cluster III fragment only bound to the anti-LRP monoclonal antibody alpha(2)MRalpha3 and weakly to RAP. Except for t-PA, we found that each of the ligands tested binds both to cluster II and to cluster IV. The affinity rate constants of ligand binding to clusters II and IV and to LRP were measured, showing that clusters II and IV display only minor differences in ligand-binding kinetics. Furthermore, we demonstrate that the subdomains C3-C7 of cluster II are essential for binding of ligands and that this segment partially overlaps with a RAP-binding site on cluster II. Finally, we show that one RAP molecule can bind to different clusters simultaneously, supporting a model in which RAP binding to LRP induces a conformational change in the receptor that is incompatible with ligand binding.

Binding Sites↗
PubMed1999

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.

Adaptor Proteins, Vesicular Transport↗
PubMed1999

Apolipoprotein CIII from guinea pig (Cavia porcellus) is shorter and less homologous than apolipoprotein CIII from other mammals.

Apolipoprotein (apo) CIII plays an important role in metabolism of triglyceride-rich lipoproteins as a regulator of lipolysis and/or lipoprotein-receptor interaction. With the method of RT-PCR, the cDNA of guinea pig apo CIII was cloned and sequenced. The deduced amino acid sequence of 91 amino acids residues consists of a highly conserved signal peptide of 20 residues and a mature protein of 71 residues. Compared to mouse, rat, dog, bovine and human apo CIII, guinea pig apo CIII has a deletion of eight or nine amino acids at its C-terminus and it shows the lowest degree of homology to the presently known apo CIII sequences. Interestingly, the most conserved areas of guinea pig apo CIII are found in two regions, residues 16-33 and residues 50-69. Corresponding regions in human and dog apo CIII were previously predicted to form amphipathic helices, which are assumed to play important roles in the inhibition of lipoprotein lipase (LPL) and binding to lipid. Our present study could be helpful for the future elucidation of the structure-function relationships and evolution of apo CIII.

Amino Acid Sequence↗
PubMed1999

Chylomicron metabolism in an animal model for hyperlipoproteinemia type I.

Mink homozygous for the mutation Pro214Leu in lipoprotein lipase (LPL) had only traces of LPL activity but amounts of LPL protein in their tissues similar to those of normal mink. In normal mink, lymph chylomicrons from rats given [3H]retinol (incorporated into retinyl esters, providing a core label) and [14C]oleic acid (incorporated mainly in triglycerides (TG)) were rapidly cleared from the circulation. In the homozygous mink, clearance was much retarded. The ratio of TG to core label in plasma did not decrease and much less [14C]oleic acid appeared in plasma. Still, half of the labeled material disappeared from the circulating blood within 30;-40 min and the calculated total turnover of TG in the hypertriglyceridemic mink was almost as large as in normal mink. The core label was distributed to the same tissues in hypertriglyceridemic mink as in normal mink. Half to two-thirds of the cleared core label was in the liver. The large difference was that in the hypertriglyceridemic mink, TG label (about 40% of the total amount removed) followed the core label to the liver and there was no preferential uptake of TG over core label in adipose or muscle tissue. In normal mink, only small amounts of TG label (<10%) appeared in the liver, while most was in adipose and muscle tissues. Apolipoprotein B-48 dominated in the accumulated TG-rich lipoproteins in blood of hypertriglyceridemic mink, even in fasted animals.

Amino Acid Substitution↗
PubMed1998

Mild oxidation of lipoproteins increases their affinity for surfaces covered by heparan sulfate and lipoprotein lipase.

Lipoprotein lipase (LPL) is present in cells involved in development of atherosclerosis (endothelial cells, smooth muscle cells, and macrophages). A direct involvement of LPL in atherogenesis has been suggested. Previously we used the surface plasmon resonance technique to study the interaction of lipoproteins with surfaces covered by heparan sulfate proteoglycans (HSPG) and LPL [A. Lookene et al. (1997) Biochemistry 36, 5267-5275]. The binding was much increased by the presence of LPL. Here we demonstrate that mild oxidation of low-density-lipoprotein (LDL) and very-low-density lipoprotein (VLDL) in vitro increases their binding to surfaces covered by HSPG and LPL, while extensive oxidation decreases it. Similar results were obtained with a lipid emulsion (Intralipid), indicating that oxidation-induced changes of the lipid part could explain the effects. LPL increased binding and uptake of the mildly oxidized (compared to nonoxidized) LDL by THP-I monocyte-derived macrophages. Our studies indicate that LPL has the highest affinity for mildly oxidized LDL and support its involvement in development of atherosclerosis.

Animals↗