Monoacylglycerol accumulation in low and high density lipoproteins during the hydrolysis of very low density lipoprotein triacylglycerol by lipoprotein lipase.

We have demonstrated that low and high density lipoproteins from monkey plasma are capable of accepting and accumulating monoacylglycerol that is formed by the action of lipoprotein lipase on monkey lymph very low density lipoproteins. Furthermore, the monoacylglycerol that accumulates in both low and high density lipoproteins is not susceptible to further hydrolysis by lipoprotein lipase but is readily degraded by the monoacylglycerol acyltransferase of monkey liver plasma membranes. These observations suggest a new mechanism for monoacylglycerol transfer from triacylglycerol rich lipoproteins to other lipoproteins. In addition, the finding that monoacylglycerol bound to low and high density lipoprotein is degraded by the liver enzyme but not lipoprotein lipase lends support to the hypothesis that there are distinct and consecutive extrahepatic and hepatic stages in the metabolism of triacylglycerol in plasma lipoproteins.     

Identification of nascent high density lipoproteins containing arginine-rich protein in human plasma.

A high density lipoprotein fraction accumulates in the plasma of patients with alcoholic hepatitis when a severe lecithin:cholesterol acyltransferase (EC 2.3.1.43) deficiency is present. The major apoprotein present in this fraction is arginine-rich protein, the fraction is a preferred substrate for lecithin:cholesterol acyltransferase, and by electron microscopy appears as stacked bilayer discs. It is proposed that the lipoprotein represents the accumulation of nascent high density lipoprotein and is the principal pathway through which arginine-rich protein is secreted by the liver in man. The results also suggest that apoprotein AI is acquired by normal high density lipoprotein during the course of lipoprotein metabolism.     

In vivo study of free and esterified cholesterol turnover in various tissues of the rat.

Rats were infused for 3.5 to 10 hrs with either red cells or plasma previously labelled in vivo by [3H]-cholesterol. Cholesterol specific radioactivities were measured in plasma, HDL, LDL and VLDL, and various tissues. Red cell infusions led to a higher labelling of free than of esterified cholesterol in the plasma of infused rats. The opposite situation was observed following plasma infusion. Comparison of free and esterified cholesterol specific radioactivities in each tissue showed that esterified cholesterol was transferred from plasma to all the tissues, except the adrenals. Study of the ratios of cholesterol specific radioactivities from one experimental group to the other in each tissue, made it possible to demonstrate clearly the occurence of hydrolysis within all the studied tissues except 5 of them where its existence remains uncertain (lung, heart, kidney, tendon, muscle) and of esterification in 3 tissues (adrenal, liver lung). In addition, ratios of cholesterol radioactivities (free/ester) were found to be identical in plasma and in 4 tissues, where neither hydrolysis nor esterification were detected (heart, muscle, kidney, tendon). This finding is an argument in favor of a simultaneous transport of free and esterified cholesterol from plasma into these 4 tissues and suggests that the entire lipoprotein particles can penetrate these tissues, with no specificity of one special class. In adrenal, unlike all other tissues: 1) the turnover of esterified cholesterol was achieved mostly by hydrolysis and esterification in situ; 2) a preferential lipoprotein class (LDL) was responsible for the transport of free cholesterol from the plasma.     

Role of net charge of low density lipoproteins in high affinity binding and uptake by cultured cells.

Selective modification of arginine residues of LDL by cyclohexanedione or acetylation of lysine residues of LDL deminishes their high affinity binding and internalisation by human skin fibroblast up to 50% as compared with native LDL. The enhanced negative charge of the modified LDL particles results in an accelerated electrophoretic mobility towards the anode. Neuraminidase treatment of cyclohexanedione-modified LDL and acetyllysine-LDL normalizes not only their electrophoretic mobility, but also restores more than 80% of the original binding and uptake capacity, the specificity of this effect being indicated by using fibroblasts deficient in LDL receptor and by competitive binding and internalization experiments.     

The mechanism of assimilation of constituents of chylomicrons, very low density lipoproteins and remnants - a new theory.

Purified remnant lipoproteins produced from chylomicrons $\text{in vivo}$ or $\text{in vitro}$ by the action of lipoprotein lipase (LPL) contain firmly bound LPL. The perfused rat liver removes the particulate bound LPL and triglyceride-labeled remnants at exactly the same rate, while purified chylomicrons are not removed. Once remnants are removed by the liver, they are not rereleased into the perfusate. These observations have led to the theory that the LPL attached to the remnant is the signal that allows the liver to “recognize” remnants from chylomicrons. This is followed by fusion of the particle with the cell surface and may be associated with the splitting off a low density lipoprotein particle. The remaining lipids of the remnant are further metabolized by the liver triglyceridase and the cholesterol esterase.     

Role of parenchymal and non-parenchymal rat liver cells in the uptake of cholesterolester-labeled serum lipoproteins.

Very low density lipoprotein (VLDL)-remnants, prepared by extrahepatic circulation of VLDL, labeled biosynthetically in the cholesterol (ester) moiety, were injected intravenously into rats in order to determine the relative contribution of parenchymal and non-parenchymal liver cells to the hepatic uptake of VLDL-remnant cholesterol (esters). 82.7% of the injected radioactivity is present in liver, measured 30 min after injection. The non-parenchymal liver cells contain 3.1±0.1 times the amount of radioactivity per mg cell protein as compared to parenchymal cells. The hepatic uptake of biosynthetically labeled (screened) low density lipoprotein (LDL) and high density lipoprotein (HDL) cholesterolesters amounts to 26.8% and 24.4% of the injected dose, measured 6 h after injection. The non-parenchymal cells contain 4.3±0.8 and 4.1±0.7 times the amount of radioactivity per mg cell protein as compared to parenchymal cells for LDL and HDL, respectively. It is concluded that in addition to parenchymal cells, the non-parenchymal cells play an important role in the hepatic uptake of cholesterolesters from VLDL-remnants, LDL and HDL.     

Hepatic uptake and metabolism of phosphatidylcholine associated with high density lipoproteins

Background

Phosphatidylcholine (PC) is the predominant phospholipid associated with high density lipoproteins (HDL). Although the hepatic uptake of cholesteryl esters from HDL is well characterized, much less is known about the fate of PC associated with HDL. Thus, we investigated the uptake and subsequent metabolism of HDL-PC in primary mouse hepatocytes.

Methods and results

The absence of scavenger receptor-BI resulted in a 30% decrease in cellular incorporation of [³H]PC whereas [³H]cholesteryl ether uptake was almost completely abolished. Although endocytosis is not involved in the uptake of cholesteryl esters from HDL, we demonstrate that HDL internalization accounts for 40% of HDL-PC uptake. Extracellular remodeling of HDL by secretory phospholipase A₂ significantly enhances HDL lipid uptake. HDL-PC taken up by hepatocytes is partially converted to triacylglycerols via PC-phospholipase C-mediated hydrolysis of PC and incorporation of diacylglycerol into triacylglcyerol. The formation of triacylglcerol is independent of scavenger receptor-BI and occurs in extralysosomal compartments.

Conclusions and general significance

These findings indicate that HDL-associated PC is incorporated into primary hepatocytes via a pathway that differs significantly from that of HDL-cholesteryl ester, and shows that HDL-PC is more than a framework molecule, as evidenced by its partial conversion to hepatic triacylglycerol.     

In vitro modification of the chemical composition of human plasma low density lipoproteins: effects of morphology and thermal properties

The effects of enzymatic action on human low density lipoproteins (LDL) occurring during in vitro incubation of plasma have been studied by chemical analysis, analytical ultracentrifugation, negative stain electron microscopy and X-ray small angle scattering. Chemically, the action of cholesteryl ester exchange and transfer proteins(s) (CEPT) leads to a relative increase in trigylcerides at the expense of cholesteryl esters. Morphologically, the particles maintain their characteristic features detectable by X-ray small angel scattering. Additional action of lecithin/cholesterol acyl transferase (LCAT) causes mainly a decrease in polar lipid contents and a reduction in particle size. The associated changes in the thermotropic transition were found to be strongly correlated to the triglyceride/cholesteryl ester ratio.     

Increased oxidazability of plasma low density lipoprotein from patients with coronary artery disease

Oxidative modification of lipoproteins may play a crucial role in the pathogenesis of atherosclerosis. This study was designed to examine whether increased lipid peroxides and/or oxidative susceptibility of plasma lipoproteins occur in patients with coronary artery disease. The levels of lipid peroxides, estimated as thiobarbituric acid-reactive substances (TBARS), were significantly greater in the plasma and very low density lipoprotein (VLDL) of symptomatic patients with coronary artery disease than in those of healthy persons, but the TBARS levels of low density lipoprotein (LDL) and high density lipoprotein (HDL) showed insignificant difference between patients and normals. To evaluate the oxidative susceptibility of lipoproteins, we employed in vitro Cu²⁺ oxidation of lipoproteins monitored by changes in fluorescenece, TBARS level, trinitrobenzene sulfonic acid (TNBS) reactivity, apolipoprotein immunoreactivity and agarose gel electrophoretic mobility. While VLDL and LDL of normal controls were oxidazed at 5–10 μM Cu²⁺, pooled VLDL and LDL of patients with coronary artery disease were oxidized at 1–2.5 μM Cu²⁺, i.e., at relatively lowver oxidative stress. At 5 μM Cu²⁺, VLDL and LDL of patients with coronary artery disease still showed at faster oxidation rate, judged by the rate of fluorescence increase, higher TBARS level, less TNBS reactivity, greater change in apo B immunoreactivity and higher electrophoretic mobility than those of normal controls. However, the difference on the oxidizability of HDL was insignificant for patients vs. normals. In conclusion, we have shown that plasm VLDL and LDL of patients with coronary artery disease are more susceptible to in vitro oxidative modification than those of health persons. The data suggest that enhanced oxidizability of plasma lipoproteins may be important factor influencing the development of coronary artery disease.     

Effect in vitro of apolipoprotein A-V on the structure and functions of recombinant high density lipoprotein

The neighboring position of apolipoprotein A-I (apoA-I) and apolipoprotein A-V (apoA-V) gene and the modulation of apoA-V on the concentrations, size and maturation of high density lipoprotein (HDL) may indicate a special relationship between apoA-V and HDL. To assess the effects of apoA-V on HDL structure and related functions in vitro, a series of recombinant HDL (rHDL) were synthesized in vitro with various mass ratios of recombinant apoA-I: apoA-V. An increase in apoA-V in rHDL resulted in enhanced lipid-binding ability, increased phospholipid content and larger particle size. Furthermore, the lipid-free and lipid-bound apoA-V in rHDL showed antioxidant capacity against low density lipoprotein (LDL) in vitro. In THP-1 derived macrophages, apoA-V of rHDL was shown to have no influence on the uptake of oxidized LDL (oxLDL) and intracellular lipid accumulation. Thus, the addition of apoA-V to rHDL resulted in changes in several rHDL properties, including increased lipid-binding ability, phospholipid content, particle size and antioxidant capacity. These alterations may explain the modulation of apoA-V on HDL in vivo and the beneficial functions of apoA-V on atherosclerosis.     

Dehydroepiandrosterone fatty acyl esters in high density lipoprotein: interaction with human vascular endothelial cells and vascular responses ex vivo

Dehydroepiandrosterone (DHEA) fatty acyl esters once incorporated in high density lipoprotein (HDL) induce a stronger vasodilatory response in rat mesenteric arteries ex vivo compared to native HDL. We studied the role of HDL receptor, scavenger receptor class B, type 1 (SR-B1), as well as estrogen and androgen receptors in the vasodilatory response of HDL-associated DHEA fatty acyl esters. Using cultured human vascular endothelial cells (HUVEC), we investigated the possible internalization and cellular response of HDL-associated DHEA esters. We prepared DHEA ester-enriched HDL by incubating human plasma in the presence of DHEA. After isolation and purification, HDL was added in cumulative doses to arterial rings precontracted with noradrenaline. Inhibition of the function of SR-B1 almost completely abolished maximal vasorelaxation by DHEA-enriched HDL while estrogen or androgen receptor blockage had no significant effect. When HUVECs were incubated in the presence of [³H]DHEA ester-enriched HDL, the amount of intracellular [³H]-radioactivity increased steadily during 24 h. Blocking of SR-B1 reduced this uptake by a mean of 30%. The proportion of unesterified [³H]DHEA, as analyzed by thin-layer chromatography, increased intracellularly and in the cell culture media after several hours of incubation of the cells in the presence of [³H]DHEA ester-enriched HDL. This indicated slow hydrolysis of DHEA fatty acyl esters and subsequent excretion of unesterified DHEA by the cells. In conclusion, DHEA-enriched HDL induced vasorelaxation via the SR-B1-facilitated pathway. However, this vasodilation is not likely to be attributed to rapid hydrolysis of HDL-associated DHEA esters by the vascular endothelium.