Kinetic analysis of apolipoproteins in postprandial hypertriglyceridaemia rabbits.

The postprandial hypertriglyceridaemia (PHT) rabbit, developed as a new animal model of metabolic syndrome, is characterized by PHT, central obesity and glucose intolerance. For detailed investigation of lipid metabolism characteristics in PHT rabbit, the plasma levels of apolipoproteins A-I, B, C-II, C-III and E were measured. Movements of apolipoproteins B100 and B48 were investigated using sodium dodecyl sulphate-polyacrylamide gel electrophoresis to determine whether postprandially increased triglyceride is exogenous or endogenous. The level of apolipoproteins A-I, B, C-II and E were increased in PHT rabbit after feeding. Apolipoproteins B100 and B48 were detected in the plasma fraction of d < 1.006 g/mL of the PHT rabbit. The postprandial increase in apolipoprotein B in the PHT rabbit reflects a numerical increase in lipoprotein particles in the blood; the increase in apolipoproteins C-II and E suggests some disturbance in lipoprotein catabolism. Apolipoprotein B48 was detected postprandially in PHT rabbits. These results suggest that delayed catabolism of exogenous lipids caused the retention of chylomicron remnants in the blood. Results also suggest that activities of the lipolytic enzyme lipoprotein lipase and hepatic triglyceride lipase were deficient and that the hepatic uptake of exogenous lipoproteins was delayed in the PHT rabbit. Especially, for examining remnant hyperlipoproteinaemia in humans, PHT rabbit is an excellent animal model for hypertriglyceridaemia research.

Diminished macrophage cholesterol removal rate by the altered HDL metabolism in the Nagase analbuminemic rat.

Dyslipoproteinemia of the Nagase analbuminemic rat (NAR) is characterized by elevated concentrations of VLDL and LDL attributed to increased rates of liver lipoprotein synthesis. Increased lysophosphatidylcholine (LPC) in NAR HDL has been attributed to high plasma LCAT activity. We show here that, as compared with Sprague-Dawley rats (SDR), NAR plasma triacylglycerol (TAG), total cholesterol (TC), HDL TAG, protein, total phospholipids (PL), LPC, and PS are increased. These alterations rendered the NAR HDL particle more susceptible to the activity of the enzyme hepatic lipoprotein lipase (HL), which otherwise was unaltered in our study. Fractional catabolic rates in blood of the autologous 125I-apoHDL (median and lower quartile values), were, respectively, 0.231 and 1.645 (n = 10) in NAR as compared with 0.140 and 0.109 (n = 10) in SDR (P = 0.012), corresponding to synthesis rates of HDL protein of 89.8 +/- 33.7 mg/d in NAR and 17.4 +/- 6.5 mg/d in SDR (P = 0.0122). Furthermore, Swiss mouse macrophage free-cholesterol (FC) efflux rates, measured as the percent [14C]-cholesterol efflux/6 h, were 8.2 +/- 2.3 (n = 9) in NAR HDL and 11.2 +/- 3.2 (n = 10) in SDR HDL (P = 0.03). Therefore, in NAR the modification of the HDL composition slows down the cell FC efflux rate, and together with the increased rate of plasma HDL metabolism influences the reverse cholesterol transport system.

Interaction of fibrin(ogen) with apolipoprotein(a): further characterization and identification of a novel lysine-dependent apolipoprotein(a)-binding site within the gamma chain 287-411 region.

Interaction of lipoprotein(a) with fibrin associated with atherosclerotic lesions promotes its accumulation in the lesions, thereby contributing to the development of atherothrombosis. Numerous studies revealed that this interaction occurs through the apolipoprotein(a) [apo(a)] component of lipoprotein(a) and COOH-terminal Lys residues generated by partial degradation of fibrin with plasmin (a COOH-Lys-dependent mechanism). At the same time, the mechanism of the interaction of apo(a) with intact fibrin(ogen) remained unclear. Our recent study identified the Lys-independent apo(a)-binding sites within the fibrin(ogen) alphaC domains which contribute to an alternative Lys-independent mechanism. In this study, we performed direct measurements of the interaction between apo(a) and various fibrin(ogen) fragments representing the whole fibrin(ogen) molecule except the alphaC regions. The experiments revealed that the apo(a)-binding site, identified previously within fibrinogen gamma chain residues 207-235 [Klose, R., et al. (2000) J. Biol. Chem. 275, 38206-38212], is a high-affinity site and mainly Lys-independent, suggesting that it should also contribute to the Lys-independent mechanism. The experiments also identified a novel Lys-dependent high-affinity apo(a)-binding site within the sequence of gamma chain residues 287-411. This site may provide interaction of apo(a) with intact fibrin(ogen) through another alternative mechanism, which depends on internal Lys residues. Thus, apo(a) may interact with intact fibrin through the Lys-independent and Lys-dependent mechanisms, while the COOH-Lys-dependent mechanism may prevail in the presence of fibrinolytic activity.

Apolipoprotein-mediated transport of nanoparticle-bound drugs across the blood-brain barrier.

Recent studies have shown that drugs that are normally unable to cross the blood-brain barrier (BBB) following intravenous injection can be transported across this barrier by binding to poly(butyl cyanoacrylate) nanoparticles and coating with polysorbate 80. However, the mechanism of this transport so far was not known. In the present paper, the possible involvement of apolipoproteins in the transport of nanoparticle-bound drugs into the brain is investigated. Poly(butyl cyanoacrylate) nanoparticles loaded with the hexapeptide dalargin were coated with the apolipoproteins AII, B, CII, E, or J without or after precoating with polysorbate 80. In addition, loperamide-loaded nanoparticles were coated with apolipoprotein E alone or again after precoating with polysorbate 80. After intravenous injection to ICR mice the antinociceptive threshold was measured by the tail flick test. Furthermore, the antinociceptive threshold of polysorbate 80-coated dalargin-loaded nanoparticles was determined in ApoEtm1Unc and C57BL/6J mice. The results show that only dalargin or loperamide-loaded nanoparticles coated with polysorbate 80 and/or with apolipoprotein B or E were able to achieve an antinociceptive effect. This effect was significantly higher after polysorbate-precoating and apolipoprotein B or E-overcoating. With the apolipoprotein E-deficient ApoEtm1Unc mice the antinociceptive effect was considerably reduced in comparison to the C57BL/6J mice. These results suggest that apolipoproteins B and E are involved in the mediation of the transport of drugs bound to poly(butyl cyanoacrylate) nanoparticles across the BBB. Polysorbate 80-coated nanoparticles adsorb these apolipoproteins from the blood after injection and thus seem to mimic lipoprotein particles that could be taken up by the brain capillary endothelial cells via receptor-mediated endocytosis. Bound drugs then may be further transported into the brain by diffusion following release within the endothelial cells or, alternatively, by transcytosis.

Lipophorin of lower density is formed during immune responses in the lepidopteran insect Galleria mellonella.

Injection of heat-killed bacteria into larvae of the greater wax moth Galleria mellonella is followed by changes in lipoprotein composition in the hemolymph. Density gradient centrifugation experiments revealed that within the first four hours after injection, a part of larval lipoprotein, high-density lipophorin (HDLp), was converted into a lipoprotein of lower density. SDS-polyacrylamide gel electrophoresis analysis of the gradient fractions and sequencing of protein fragments, established that the exchangeable apolipoprotein apolipophorin III (apoLp-III), a potent immune-activator, was associated with this newly formed lipophorin. To investigate further the influence of lipophorin-associated apoLp-III on immune-related reactions, we performed in vitro studies with isolated hemocytes from G. mellonella and lipophorins from the sphinx moth Manduca sexta, as a natural source of high amounts of low-density lipophorin (LDLp) and HDLp. The hemocytes were activated to form superoxide radicals upon incubation with LDLp, but not with HDLp. Fluorescence-labeled LDLp was specifically taken up by granular cells. This process was inhibited by adding an excess of unlabeled LDLp, but not by HDLp. We hypothesize that larval lipophorin formed in vivo is an endogenous signal for immune activation, specifically mediated by the binding of lipid-associated apoLp-III to hemocyte membrane receptors.

Plasma kinetic behavior in hyperlipidemic subjects of a lipidic microemulsion that binds to low density lipoprotein receptors.

It was previously reported that a protein-free microemulsion (LDE) with structure roughly resembling that of the lipid portion of low density lipoprotein (LDL) was presumably taken up by LDL receptors when injected into the bloodstream. In contact with plasma, LDE acquires apolipoproteins (apo) including apo E that would be the ligand for receptor binding. Currently, apo were associated to LDE by incubation with high density lipoprotein (HDL). LDE-apo uptake by mononuclear cells showed a saturation kinetics, with an apparent K(m) of 13.1 ng protein/mL. LDE-apo is able to displace LDL uptake by mononuclear cells with a Ki of 11.5 ng protein/mL. LDE without apo is, however, unable to displace LDL. The uptake of 14C-HDL is not dislocated by increasing amounts of LDE-apo, indicating that HDL and LDE-apo do not bind to the same receptor sites. In human hyperlipidemias, LDE labeled with 14C-cholesteryl ester behaved kinetically as expected for native LDL. LDE plasma disappearance curve obtained from eight hypercholesterolemic patients was markedly slower than that from 10 control normolipidemic subjects [fractional clearance rate (FCR) = 0.02 +/- 0.01 and 0.12 +/- 0.04 h-1, respectively; P < 0.0001]. On the other hand, in four severely hypertriglyceridemic patients, LDE FCR was not significantly different from the controls (0.07 +/- 0.03 h-1). These results suggest that LDE can be a useful device to study lipoprotein metabolism.

Functional and metabolic differences between elastase-generated fragments of human lipoprotein[a] and apolipoprotein[a].

We have previously shown that a functional free apolipoprotein[a] (apo[a]) can be isolated from its parent lipoprotein[a] (Lp[a]) by a mild reductive procedure. To shed further light on the properties of Lp[a] and apo[a] we subjected them to a limited proteolysis by porcine pancreatic elastase. This enzyme cleaved both at the Ile3520-Leu3521 bond in the linker between kringles IV-4 and IV-5 of apo[a] generating two fragments F1 and F2. In contrast to F1, which represented the N-terminal portion of apo[a] and was functionally inert, F2, representing the C-terminal domain, bound to lysine-Sepharose, fibrinogen, and fibronectin and formed a miniLp[a] particle when incubated with LDL. The proteolytic pattern by pancreatic elastase was also exhibited by human leukocyte elastase. F1, injected intravenously into normal mice, was rapidly cleared (Ty2, 2.9 h) and after 1 h fragments in the size range of 100-33 kDa were observed in the urine. In turn, F2 had a longer residence time (Ty2, 5 h) and was excreted in the urine only after 5 h as fragments of 70-45 kDa. Fragments in the same size range as found after F1 injection were also present in the urine after injection of apo[a] or Lp[a]. Moreover, apo[a] fragments of the size seen in mouse urine were spontaneously present in normal human urine and appeared derived from larger apo[a] fragments in the plasma. Our results indicate that enzymes of the elastase family cleave human apo[a] in vitro into two main fragments that differ in structural and functional properties and metabolic behavior. The comparable size of apo[a] fragments observed in the urine of humans and injected mice invites the speculation that enzymes of the elastase family may play a role in the biology of Lp[a] in vivo.

[The role of apolipoproteins in lipid metabolism]. / Die Rolle der Apolipoproteine für den Lipidstoffwechsel.

There is little doubt today that apolipoproteins play a key role in lipid metabolism and thus in atherogenesis. There are five major classes of apo Lp known: Apo AI, the main component of HDL not only mediates the action of LCAT, a key enzyme in cholesterol metabolism, but also through specific cell receptors is responsible for the reverse cholesterol transport, which is discussed as the main atherogenic process. Apo B is necessary for the secretion of neutral lipids out of the liver and the intestine. In addition, apo B containing lipoproteins are recognized by specific cell surface receptors leading to the fast removal of cholesterol rich fractions from circulation. Apo C proteins regulate the activity of lipoprotein lipase, the key enzyme of triglyceride metabolism. Apo E containing lipoproteins are recognized by the B/E-receptor with a 10 to 100 fold affinity. There exists, however, another specific receptor for Apo E, which is responsible for the fast removal of the atherogenic remnants from circulation. Apo E in addition serves to secrete deposited cholesterol out of macrophages and foam cells. Apolipoprotein(a) is a peculiar fraction of apo B containing lipoproteins whose biological function is completely unknown. Cloning of the cDNA revealed striking similarities of apo(a) with the structure of plasminogen. The cross connection of Lp(a) with hemostasis and thrombogenesis is currently focus of intensive research. The knowledge of the specific function of apolipoproteins in lipid metabolism arose to a great extent from the characterization of apo-Lp isoforms and their impact of atherogenesis. In addition, intensive research by molecular biology techniques helped to unravel the pathophysiology in a wide array.

Behavior of human apolipoprotein A-I: phospholipid and apoHDL:phospholipid complexes in vitro and after injection into rabbits.

Apolipoprotein A-I was purified from human high density lipoprotein and complexed with polyunsaturated phosphatidylcholine (PC) in deoxycholate (Lipostabil); the bile salt was removed subsequently by dialysis. The behavior of the resultant apoA-I/PC complexes was compared with that of Lipostabil in vitro and after injection into rabbits. In vivo apoA-I/PC complexes had the density of HDL throughout but had both alpha and pre beta electrophoretic mobility, the latter probably reflecting dissociation of apoA-I from PC. Lipostabil initially behaved like LDL but gradually acquired the density of HDL after incubation with plasma and in vivo. Both preparations increased plasma total phospholipids in normolipidemic rabbits to a similar extent, but, increments in HDL phospholipid were greater after apoA-I/PC complexes were injected. ApoHDL/PC complexes, prepared in a similar manner, appeared to promote efflux of cholesterol from perfused rabbit aortas in the presence of lecithin:cholesterol acyltransferase (LCAT) activity, consistent with a stimulatory effect on cholesterol mobilization. Injection of apoHDL/PC complexes into hyperlipidemic rabbits decreased plasma cholesterol but increased HDL cholesterol, whereas Lipostabil decreased both. These findings suggest that human apoA-I/PC complexes resemble HDL in their behavior more closely than does Lipostabil, and show that both types of liposome undergo modification upon interaction with plasma. It remains to be shown whether they possess any therapeutic potential.