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.     

Ubiquitination regulates the assembly of VLDL in HepG2 cells and is the committing step of the apoB-100 ERAD pathway

Apolipoprotein B-100 (apoB-100) is degraded by endoplasmic reticulum-associated degradation (ERAD) when lipid availability limits assembly of VLDLs. The ubiquitin ligase gp78 and the AAA-ATPase p97 have been implicated in the proteasomal degradation of apoB-100. To study the relationship between ERAD and VLDL assembly, we used small interfering RNA (siRNA) to reduce gp78 expression in HepG2 cells. Reduction of gp78 decreased apoB-100 ubiquitination and cytosolic apoB-ubiquitin conjugates. Radiolabeling studies revealed that gp78 knockdown increased secretion of newly synthesized apoB-100 and, unexpectedly, enhanced VLDL assembly, as the shift in apoB-100 density in gp78-reduced cells was accompanied by increased triacylglycerol (TG) secretion. To explore the mechanisms by which gp78 reduction might enhance VLDL assembly, we compared the effects of gp78 knockdown with those of U0126, a mitogen-activated protein kinase/ERK kinase1/2 inhibitor that enhances apoB-100 secretion in HepG2 cells. U0126 treatment increased secretion of both apoB100 and TG and decreased the ubiquitination and cellular accumu-lation of apoB-100. Furthermore, p97 knockdown caused apoB-100 to accumulate in the cell, but if gp78 was concomitantly reduced or assembly was enhanced by U0126 treatment, cellular apoB-100 returned toward baseline. This indicates that ubiquitination commits apoB-100 to p97-mediated retrotranslocation during ERAD. Thus, decreasing ubiquitination of apoB-100 enhances VLDL assembly, whereas improving apoB-100 lipidation decreases its ubiquitination, suggesting that ubiquitination has a regulatory role in VLDL assembly.     

Effects of apolipoprotein B-100 on the metabolism of a lipid microemulsion model in rats

In previous studies, it was shown that lipid microemulsions resembling LDL (LDE) but not containing protein, acquire apolipoprotein E when injected into the bloodstream and bind to LDL receptors (LDLR) using this protein as ligand. Aiming to evaluate the effects of apolipoprotein (apo) B-100 on the catabolism of these microemulsions, LDE with incorporated apo B-100 (LDE-apoB) and native LDL, all labeled with radioactive lipids were studied after intraarterial injection into Wistar rats. Plasma decay curves of the labels were determined in samples collected over 10 h and tissue uptake was assayed from organs excised from the animals sacrificed 24 h after injection. LDE-apo B had a fractional clearance rate (FCR) similar to native LDL (0.40 and 0.33, respectively) but both had FCR pronouncedly smaller than LDE (0.56, P<0.01). Liver was the main uptake site for LDE, LDE-apoB, and native LDL, but LDE-apoB and native LDL had lower hepatic uptake rates than LDE. Pre-treatment of the rats with 17α-ethinylestradiol, known to upregulate LDLR, accelerated the removal from plasma of both LDE and LDE-apoB, but the effect was greater upon LDE than LDE-apoB. These differences in metabolic behavior documented in vivo can be interpreted by the lower affinity of LDLR for apo B-100 than for apo E, demonstrated in in vitro studies. Therefore, our study shows in vivo that, in comparison with apo E, apo B is a less efficient ligand to remove lipid particles such as microemulsions or lipoproteins from the intravascular compartment.     

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.     

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.     

Presence of B-100 in rat mesenteric chyle

Molecular forms of apolipoprotein B (ApoB) were studied in the rat intestinal chyle by SDS-polyacrylamide gel electrophoresis, immunoblotting and immunodiffusion. Time studies on intestinal chyle showed the presence of B-100 in all the samples analyzed within 3 hr after drawing. However, the analyses repeated on day 2 or day 3 revealed disappearance of B-100 and appearance of B-48. Addition of 3 mM EDTA, 10 mM diisopropylfluorophosphate, 5 mM chloroquine and 10 mM epsilon-amino caproic acid slowed down but could not prevent the disappearance of B-100. Chylomicrons isolated from chyle in the presence of preservatives immediately after drawing displayed B-100 as a major and B-48 as a minor ApoB form. However, repeatedly washed chylomicrons or those isolated from chyle 18-24 hr after drawing showed B-48 as the only ApoB present. These results suggest that rat intestine synthesizes B-100 which is quickly converted to smaller molecular form.     

CD36 is a receptor for oxidized high density lipoprotein: implications for the development of atherosclerosis

Atherosclerotic plaques result from the excessive deposition of cholesterol esters derived from lipoproteins and lipoprotein fragments. Tissue macrophage within the intimal space of major arterial vessels have been shown to play an important role in this process. We demonstrate in a transfection system using two human cell lines that the macrophage scavenger receptor CD36 selectively elicited lipid uptake from Cu(2+)-oxidized high density lipoprotein (HDL) but not from native HDL or low density lipoprotein (LDL). The uptake of oxHDL displayed morphological and biochemical similarities with the CD36-dependent uptake of oxidized LDL. CD36-mediated uptake of oxidized HDL by macrophage may therefore contribute to atheroma formation.     

Selective effects of hydrocortisone on intestinal lipoprotein and apolipoprotein synthesis in the human fetus

Studies employing human fetal intestine have yielded much interesting information on the role of polarized enterocytes in fat absorption and transport. Using the organ culture model, we examined the influence of hydrocortisone on the synthesis and secretion of lipids and lipoproteins. Human jejunal explants were cultured for 5 days at 37°C in serum-free medium containing either [¹⁴C]-oleic acid or [¹⁴C]-acetate, alone or supplemented with hydrocortisone (25 or 50 ng/ml). The uptake of [¹⁴C]-oleic acid was associated with the production of triglycerides, phospholipids, and cholesteryl esters, which were all affected by hydrocortisone. This hormonal agent (50 μg) led to the marked reduction of secreted triglycerides (43%, P < 0.01), phospholipids (39%, P < 0.01), and cholesteryl esters (36%, P < 0.05) without altering the characteristic distribution of tissue and medium lipid classes. Similarly, hydrocortisone significantly (P < 0.01) decreased (∼60%) the incorporation of [¹⁴C]-acetate into secreted free and esterified cholesterol in the medium. With [¹⁴C]-oleic acid as a precursor, hydrocortisone significantly diminished the delivery of chylomicrons and very low density lipoproteins to the medium while consistently enhancing the secretion of high density lipoproteins. In parallel, [³⁵S]-methionine pulse-labeling of jejunal explants revealed the concomitant inhibitory effect of hydrocortisone on apo B-100 synthesis and hydrocortisone's stimulatory effect on apo B-48 and apo A-I. These studies suggest that glucocorticoids play a critical role in lipoprotein processing during intestinal development. J. Cell. Biochem. 66:65–76 1997. © 1997 Wiley-Liss, Inc.     

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.     

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.     

Effects of microtubule-disruptive and membrane-stabilizing agents on low density lipoprotein metabolism by cultured human fibroblasts

The cellular mechanisms involved in the uptake and metabolism of low density lipoprotein (LDL) by cultured normal human fibroblasts have been investigated with the aid of drugs known to disrupt cytoplasmic microtubules or to inhibit membrane fusion.Two drugs which disrupt microtubules by differing mechanisms, colchicine and vinblastine, each reduced the high affinity surface binding of ¹²⁵I-labelled LDL by fibroblasts. Associated reductions of the endocytosis and degradation of the lipoprotein could be attributed almost entirely to this effect. In contrast, lumicolchicine, an analogue of colchicine without microtubule-disruptive activity, had little or no effect on ¹²⁵I-labelled LDL metabolism.Each of two groups of membrane-stabilizing agents, the phenothiazines and the tertiary amine local anaesthetics, directly inhibited both the internalization of ¹²⁵I-labelled LDL following high affinity binding to cell surface receptors and the catabolism of the lipoprotein subsequent to endocytosis, supporting previous morphological evidence for the importance of membrane fusion in these processes.     

Increased cellular cholesterol efflux in glycogen storage disease type Ia mice: a potential mechanism that protects against premature atherosclerosis

Glycogen storage disease type Ia (GSD-Ia) patients manifest a pro-atherogenic lipid profile but are not at elevated risk for developing atherosclerosis. Serum phospholipid, which correlates positively with the scavenger receptor class B type I (SR-BI)-mediated cholesterol efflux, and apolipoprotein A-IV and E, acceptors for ATP-binding cassette transporter A1 (ABCA1)-mediated cholesterol transport, are increased in GSD-Ia mice. Importantly, sera from GSD-Ia mice are more efficient than sera from control littermates in promoting SR-BI- and ABCA1-mediated cholesterol effluxes. As the first step in reverse cholesterol transport, essential for cholesterol homeostasis, these observations provide one explanation why GSD-Ia patients are apparently protected against premature atherosclerosis.     

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.     

Critical role of cholesterol ester transfer protein in nicotinic acid-mediated HDL elevation in mice

Nicotinic acid is a commonly used anti-dyslipidemic agent that increases plasma levels of HDL-cholesterol and decrease triglycerides (TG), and VLDL- and LDL-cholesterol. The most well-studied effect of nicotinic acid is its ability to lower plasma free fatty acids, which has been observed in humans and many animal models. However, its ability to raise HDL in humans has not been replicated in animal models, which precludes studying the mechanism of HDL elevation. Here we studied lipid-modulating effects of nicotinic acid in mice carrying genomic DNA fragments that drive expression of various human genes in the mouse liver. Treatment with nicotinic acid reduced serum levels of HDL cholesterol in wild-type and human apolipoprotein B100 (apoB100)-transgenic mice. In contrast, nicotinic acid treatment of mice that express human cholesteryl ester transfer protein (CETP), with or without concomitant apoB100 expression, resulted in a significant increase of HDL cholesterol and reduction of TG, VLDL- and LDL-cholesterol. These data demonstrate a critical role of CETP in nicotinic acid-mediated HDL elevation, and suggest that mice carrying the human CETP gene may be useful animal models for studying the HDL-elevating effect of nicotinic acid.     

Monoclonal antibodies to low density lipoprotein used for the study of low- and very-low-density lipoproteins, in "ELISA" and immunoprecipitation technics

Seven monoclonal antibodies to low-density lipoprotein were studied by the ELISA for their reactivity with LDL or VLDL. Cotitration experiments showed that five of them are addressed to different antigenic epitopes. Two of the monoclonal antibodies were temperature independent whereas the others had a decreased binding activity at 37 degrees C compared to that obtained at 25 degrees C or 4 degrees C, suggesting the presence of antibodies directed to sequence or conformation epitopes, respectively. All antibodies reacted with both LDL and VLDL; four of them had a higher affinity for LDL and two others for VLDL. Immunoprecipitation of LDL and/or VLDL was observed upon immunodiffusion with certain pairs of antibodies. This may allow the use of pairs of monoclonal antibodies to LDL for the quantitative determination of apolipoprotein B in serum LDL and VLDL.