Olga Stein

The incorporation and disappearance of fatty acids in the rat epididymal fat pad studied by the in vivo incubation technique

Abstract Using the in vivo incubation technique it has been shown that the rat epididymal fat pad incubated in a mixture of two fatty acids incorporates palmitic acid slightly faster than oleic or linoleic acid and considerably more rapidly than stearic acid. Fasting induces mobilization of palmitic, oleic and linoleic acids at the same rate, with conservation of the adipose-tissue fatty acid composition. During 60 days no measurable saturation of linoleic to oleic or stearic acids occurred. The half life of palmitic and linoleic acids in the epididymal fat pad has been estimated as 163 and 187 days, respectively. Comparable values have been obtained when the disappearance rate of [ 14 C]palmitic acid from the epididymal fat pad has been measured following a single oral dose.

Comparative Uptake of Rat and Human Serum Low-Density and High-Density Lipoproteins by Rat Aortic Smooth Muscle Cells in Culture

Rat aortic smooth muscle cells in culture were exposed to rat and human serum low-(LDL) and high-density (HDL) lipoproteins labeled with 125I. 125I-lipid was taken up preferentially from all of the lipoproteins used. 125I-protein uptake of both rat LDL and HDL was significantly higher than that of the corresponding human lipoproteins, and human LDL was preferred to human HDL. The uptake of delipidated high-density apolipoproteins of either rat or human origin was very low. About 3–4% of the interiorized rat LDL and HDL was catabolized during 48 hours of incubation. On electron microscopic autoradiography of cells incubated with rat 125I-LDL, the concentration of label, representing mainly 125I-protein, was associated with secondary lysosomes. These results suggest that, if the protein uptake represents particle uptake, the preferential uptake of human LDL compared with human HDL could account in part for the finding that LDL acts as a more potent feedback suppressor of 3-hydroxy-3-methylglutaryl CoA reductase than does HDL.

Metabolic activity of rat epididymal fat pad labeled selectively by an in vivo incubation technique

Abstract A method of in vivo incubation of rat epididymal fat pads for selective labeling of this tissue is described. The effects of buffer, fatty acid concentration, time, glucose and hormones on the incorporation of [I−14C]palmitic acid were inveestigated. These studies revealed the presence of two glyceride compartments in adipose tissue: a small one, located in the cytoplasmic particulate fraction, with a half-life of 20–25 min, and a large slowly turning over compartment containing most of the glycerides in the fat cell. After 20 min of in vivo incubation of the epididymal fat pad a diglyceride of high specific activity can be separated from the labeled triglyceride. It was also shown that newly deposited fat mixes rapidly with the pre-existing fat of the adipose cell.

Use of 3H-cholesteryl linoleyl ether for the quantitation of plasma cholesteryl ester influx into the aortic wall in hypercholesterolemic rabbits.

In this study use was made of 3 H-cholesteryl linoleyl ether (CLE), a nondegradable analogue of cholesteryi ester (CE) to measure plasma lipoprotein CE influx into rabbit aorta. Autologous serum labeled with 3 H-CLE was injected into seven hypercholesterolemic rabbits, and more than 90% of the label was recovered in the plasma compartment 10 minutes after injection. Between 4 hours and 3 days the label was cleared from the circulation with a tV2 of about 24 hours. Between 4 and 24 hours the lipoproteins isolated at d < 1.006, d < 1.019, and d < 1.063 approached similar specific activity, assuming that 3 H-CLE had mixed with the lipoprotein CE pool. The rabbits were killed 7 to 14 days after injection when plasma radioactivity decreased to < 0.03% of injected dose/ml. Total recovery of the CLE ranged from 70% to 95% and 48% to 72% were found in the liver. The minimum influx of plasma CE into the aortic Intima was determined by dividing the label found in the artery by the mean specific activity of the labeled compound in the plasma. The minimum influx into regions with atheromatous involvement ranged from 0.8 to 3.4 jug CE/cm2/hr. The rate of influx was highly correlated with the amount of CE mass in the jntima and media indicating that the bulk of aortic CE is derived from plasma lipoprotein CE. The method described might be useful in distinguishing between possible effects of antiatherogenic drugs on plasma CE influx into the aortic wall from an effect on intracellular CE hydrolysis and subsequent efflux of free cholesterol from the artery.

Synthesis of ether analogs of lipoprotein lipids and their biological applications.

Publisher Summary This chapter reviews the synthesis of cholesteryl ethers, retinyl ethers, tri- and dialkyl glycerols, and dialkyl phospholipids, which are nonhydrolyzable analogs of the lipid moieties of lipoproteins. In recent years, there has been considerable interest in the use of liposomes as carriers of drugs to be delivered to specific target sites. If liposomes are to be used to deliver therapeutic compounds to cells it may be important to use phospholipids which are not readily degraded by cellular or circulating enzymes. The lipids transferred by lipoprotein lipase (LPL) to cells were localized in three compartments: trypsin releasable, resistant, and metabolic; the latter was a chloroquine-sensitive pool as evidenced by inhibition of cholesteryl ester hydrolysis. Labeled phosphatidylcholine (PC) and, to a lesser extent, dioleyl ether phosphatidylcholine (DOEPC), in the trypsin releasable pool returns to the medium, while cholesteryl linoleyl ether (CLE) and cholesteryl ester (CE) required cholesteryl ester transfer protein for release. The transfer of CLE and CE into a trypsin-resistant compartment does not require metabolic energy and occurs also in formaldehyde-fixed cells; metabolic energy is needed for the translocation of CLE and CE into the lysosomal compartment, presumably by a process of endocytosis.

Scavenger Receptor Activity Is Increased in Macrophages From Rabbits With Low Atherosclerotic Response: Studies in Normocholesterolemic High and Low Atherosclerotic Response Rabbits

We have previously described 2 strains of New Zealand White rabbits with a high (HAR) or low (LAR) atherosclerotic response to hypercholesterolemia. In the present study, we focused on class A scavenger receptor (SR-A) activity and ApoE expression in macrophages from both rabbit strains. These parameters play a crucial role in maintaining cholesterol homeostasis in the arterial wall and may be involved in the development of atherosclerosis. SR activity, as measured by uptake of DiI-labeled acetylated LDL, was significantly higher in macrophages from LAR rabbits (2177+/-253 ng/mg cell protein) than in macrophages from HAR rabbits (1153+/-200 ng/mg cell protein). The higher SR activity was caused by a greater number of SRs (apparent Vmax, 4100 ng/mg in LAR and 1980 ng/mg in HAR rabbits). The high SR activity in macrophages from LAR rabbits was associated with a significantly higher expression of SR-A mRNA compared with macrophages from HAR rabbits. However, the latter finding could not be explained by differences in the activity of transcription factor-activating protein 1 (AP-1), which was comparable in macrophages from both strains of rabbits. Because under certain circumstances SR-A mRNA expression is regulated in parallel with ApoE expression, we also evaluated this parameter. Although ApoE mRNA was 74% higher in macrophages from LAR rabbits, the difference did not reach statistical significance. In conclusion, the increased expression of SR-A in macrophages in the presence of adequate amounts of ApoE may play a role in attenuating atherosclerosis in LAR rabbits.

Metabolism of Plasma Lipoproteins

This review will attempt to survey some of the progress made in the study of chylomicron and very low density lipoprotein (VLDL) metabolism since the last International Symposium on Atherosclerosis in Tokyo, 1976. Emphasis will be given to the differences in the metabolic fate of these lipoproteins in the human and smaller experimental animals. Because of these considerations as well as space limitation, the review will be selective but not exhaustive.

Metabolism of lysolecithin by the perfused rat heart

Abstract Lysolecithin prepared from biosynthetically labeled rat-liver lecithin was complexed with either whole rat serum or bovine serum albumin and was shown to be bound mainly to the albumin fraction. Isolated rat hearts showed a rapid uptake of lysolecithin labeled either in the phosphate or fatty acid moiety, and the extent of its uptake was proportional to the ratio of lysolecithin to albumin in the perfusion medium. Autoradiography of histologie sections of hearts labeled with [14C]- or [32P]-lysolecithin showed that the radioactivity was distributed diffusely and was not confined to the blood vessels. Hearts labeled with lysolecithin were reperfused with a medium devoid of protein and no loss of radioactivity from the heart was observed. When serum or bovine albumin were added to the reperfusion medium a major part of the lysolecithin could be eluted. Simultaneous labeling of the heart with [131I]-albumin and [32P]lysolecithin showed that the latter was taken up by the heart not as an albumin-lysolecithin complex. A part of the lysolecithin taken up by the heart was acylated to lecithin, a reaction which was progressive with time of reperfusion of a pulse labeled heart. In contradistinction to lysolecithin the labeled lecithin could not be eluted into a protein containing reperfusion medium. It is postulated that binding of lysolecithin to a tissue acceptor plays a part in the uptake of this phospholipid by the heart and that the lysolecithin might be involved in cellular membrane renewal.

Effects of Intravenously Administered Poly-D L-lysine in Rats

Summary Poly-D L-lysine was administered intravenously to rats. Lethal doses of 1.5-2 mg per 100 g body weight caused a disturbance in thrombin formation, pulmonary and cardiac edema and death within 12 minutes. The high dose of 8 mg polylysine per 100 g body weight caused red cell agglutination and prolonged clotting time. Sublethal doses also caused a disturbance in thrombin formation, slight hemolysis and reticulocytosis, and transitory symptoms of respiratory distress. Heparin and synthetic poly-L-aspartic acid were antagonistic to polylysine and prevented death of polylysine-treated rats, hemolysis and coagulation disturbance. These effects in vivo corroborated the observations on the biological action of polylysine in vitro.

Smooth muscle cells and atherosclerosis

The role of smooth muscle cells in atherogenesis involves cell proliferation and accretion of cholesteryl ester. Smooth muscle cell proliferation, controlled by growth factors produced locally, contributes to progression of atheroma and to restenosis after percutaneous transluminal coronary angioplasty. Accretion of cholesteryl ester in smooth muscle cells is mediated by factors secreted by macrophages. Induction of the scavenger receptor in smooth muscle cells may promote the transformation to foam cells. Novel approaches to combat restenosis include gene transfer into smooth muscle cells using different vectors.