125I-labeled Human Low Density Lipoprotein Research Articles

Lipoprotein (a) displays increased accumulation compared with low-density lipoprotein in the murine arterial wall

Lipoprotein (a) (Lp(a)) is known to be an independent risk factor for cardiovascular disease, but the mechanisms by which it contributes to this disease remain unclear. Current evidence indicates that the closely related plasma particle, low-density lipoprotein (LDL), may initiate atherosclerosis through deposition in the arterial wall. This study has compared the ability of both lipoproteins to enter and accumulate within the arterial wall. Experiments were conducted in vivo with animals from two strains of mice: C57BL/6 mice, which develop fatty streak lesions upon challenge by a high-fat diet, and C3H/HeJ mice, which are resistant to lesion formation. Animals from both strains were maintained up to 16 weeks either on chow or high-fat diet. The mice were intravenously injected with 125I-labeled human Lp(a) or 125I-labeled human LDL in equimolar amounts and the lipoprotein allowed to circulate in vivo for 2 or 24 h. Transverse sections of the aortic root including sites of predilection for lesion formation at the commissures of the valve were prepared and examined after autoradiography. The autoradiographic grains over lesions and histologically uninvolved areas were enumerated and compared after normalization. Both Lp(a) and LDL demonstrated nearly ten times greater accumulation in lesions compared with histologically uninvolved areas from C57BL/6 mice. Analyses of histologically uninvolved areas from both strains of mice showed a significantly higher accumulation of Lp(a) than LDL. Finally, significantly higher accumulations of both Lp(a) and LDL occurred in the histologically uninvolved intima and subintima of lesion-prone C57BL/6 mice as compared with lesion-resistant C3H/HeJ mice after 5 weeks on the diets. We propose that enhanced accumulation of Lp(a) in the arterial wall accounts, in part, for the increased risk of cardiovascular disease.

Effects of Diets Rich in Fermentable Carbohydrates on Plasma Lipoprotein Levels and on Lipoprotein Catabolism in Rats

This study was conducted to examine the effects of a combination of several dietary fibers (5% guar gum, 5% apple pectin, 15% wheat bran, 22% soybean fiber) and crude potato starch (23%) on plasma lipids and lipoproteins and on liver lipid concentration in rats fed a diet containing either 15% lard or 5% oil with or without dietary cholesterol/cholic acid. Male Wistar rats ate the test diets for 3 wk. The incorporation of fiber and crude potato starch into the diet resulted in a significant enlargement of the cecum; it also increased the concentration of volatile fatty acids and the pool of acetate, propionate and butyrate. Feeding this fermentable carbohydrate decreased plasma cholesterol and triglyceride levels in rats given a low fat diet and prevented the expected rise in plasma cholesterol and triglycerides in rats fed cholesterol/cholic acid or lard. Further studies of high density lipoprotein (HDL) composition, 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMG-CoA reductase) activity and 125I-labeled human low density lipoprotein (LDL) turnover were done in the group fed the low fat diet without added cholesterol/cholic acid. The study of the HDL fraction in rats fed a diet rich in fermentable carbohydrates demonstrated a decrease in the HDL1 subpopulation and in the proportion of apolipoprotein E. Plasma clearance of intravenously injected 125I-labeled LDL was faster in rats fed this diet than in rats fed the fiber-free diet. In the liver, cholesterol and triglyceride levels were depressed whereas the activity of HMG-CoA reductase was increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Hormonal determinants of apolipoprotein B,E receptor expression in human liver. Positive association of receptor expression with plasma estrone concentration in middle-aged/elderly women

The relationships of the expression of hepatic low-density lipoprotein (LDL) receptors (apo B,E receptors) to several plasma hormone concentrations were examined in 15 fasted women aged 37–75 years (mean, 57 years), who were undergoing laparotomy for non-neoplastic disease. No subject had clinical or biochemical evidence of familial hypercholesterolemia, renal disease, hepatic disease, or endocrine disease. Hepatic apo B,E receptor expression was quantified in vitro as the EDTA-suppressible binding of 125I-labeled human LDL (15 μg protein/ml) by liver homogenate at 37°C; values were 23–75 ng LDL protein/mg cell protein (mean, 47 ng/mg). Receptor expression was strongly correlated with plasma estrone concentration ( r s = +0.70, P = 0.035), but was unrelated to the concentrations of testosterone, thyroxine, free triiodothyronine, cortisol, sex hormone-binding globulin (SHBG) or cortisol-binding globulin. Insulin and estradiol concentrations were mostly very low. The correlation of receptor expression with plasma total estrone concentration reflected associations with both the albumin-bound ( r s = +0.78, P = 0.014) and unbound ( r s = +0.80, P = 0.009) fractions, but not with the SHBG-bound fraction ( r s = −0.22, P = 0.574), of this hormone. As the non-SHBG-bound fractions of gonadal steroids are considered to be the biologically active components, these results are consistent with experimental evidence that the synthesis of apo B,E receptors in hepatocytes is stimulated by estrogens, and suggest that circulating estrone may be the major hormonal determinant of receptor expression in fasted middle-aged/elderly women.

Lipophilic beta-adrenoceptor antagonists stimulate low density lipoprotein receptor activity in human skin fibroblasts.

The effect of beta-adrenoceptor antagonists on the receptor-mediated low density lipoprotein (LDL) binding and internalization was studied in vitro in human skin fibroblasts. The cellular uptake of 125I-labeled human LDL was dose dependently elevated by some, but not all, of the drugs used. This effect of beta-adrenoceptor antagonists was positively related to their lipophilicity, and was prevented by cycloheximide and by alpha-amanitin. Scatchard analysis of the saturable LDL binding indicates an increased number of LDL binding sites. Our studies show that the stimulating effect of beta-adrenoceptor antagonists on the high affinity LDL binding and internalization in human skin fibroblasts involves DNA transcription and new protein synthesis, and identify drug lipophilicity as a major determinant of this action. This effect could be relevant in vivo in adipose tissue which accumulates lipophilic drugs and derives its cholesterol mainly from circulating LDL.

Local variation in arterial wall permeability to low density lipoprotein in normal rabbit aorta.

Normal rabbits were injected intravenously with horseradish peroxidase (HRP) and 125I-labeled human low density lipoprotein (LDL), and the aortas were perfusion-fixed. Subsequent visualization of HRP in the aortas was produced by reaction of the tissue with diaminobenzidine and hydrogen peroxide. The luminal surface of the aortas showed many small punctate foci of brown reaction product to the HRP, which represented penetration of the HRP into the vessel wall. The foci were scattered over the luminal surface, and most of the focal areas were less than 1 mm in diameter. The concentration of LDL was up to 47 times greater in these focal areas than in surrounding noncolored regions not showing increased permeability to HRP. Small circumscribed foci of heightened permeability to LDL may predispose to the local accumulation of lipid and ultimately to the formation of atherosclerotic plaques.

Evolution of low density lipoprotein structure probed with monoclonal antibodies.

To assess the relationship of apoB structures in different species of animals, the expressions of apoB epitopes in the sera or plasmas of 23 different mammalian species and one marsupial, and on the low density lipoprotein (LDL) from three species of apes, six species of monkeys, and eight non-primates were measured in competitive radioimmunoassays. The abilities of the sera or LDL to compete with 125I-labeled human LDL for binding to seven monoclonal antihuman LDL antibodies immobilized on microtiter plates were determined. LDL of apes bound to most antibodies, while monkey LDL bound to two or three antibodies. Other mammalian LDL bound only weakly to two of the antibodies or to none. The two monoclonal antibodies binding the LDL of more species were those antibodies which also inhibited the binding to and degradation of LDL by human fibroblasts. The rank order of binding of the LDL of a given species to the antibodies correlated with the rank order inhibition of binding and degradation of 125I-labeled human LDL in the human fibroblast system. This suggests that epitopes spatially located near the recognition site of apoB for cellular receptors have a greater tendency to be conserved.

Regulation of the uptake and degradation of beta-very low density lipoprotein in human monocyte macrophages.

In normal human monocyte macrophages 125I-labeled beta-migrating very low density lipoproteins (125I-beta-VLDL), isolated from the plasma of cholesterol-fed rabbits, and 125I-human low density lipoprotein (LDL) were degraded at similar rates at protein concentrations up to 50 micrograms/ml. The high affinity degradation of 125I-labeled human LDL saturated at approximately 50 micrograms/ml; however, 125I-labeled rabbit beta-VLDL high affinity degradation saturated at 100-120 micrograms/ml. The activity of the beta-VLDL receptor was 3-fold higher than LDL receptor activity on freshly isolated normal monocyte macrophages, but with time-in-culture both receptor activities decreased and were similar after several days. The degradations of both beta-VLDL and LDL were Ca2+ sensitive, were markedly down regulated by sterols, and were up regulated by preincubation of the cells in a lipoprotein-free medium. The beta-VLDL receptor is genetically distinct from the LDL receptor as indicated by its presence on monocyte macrophages from a familial hypercholesterolemic homozygote. Human thoracic duct lymph chylomicrons as well as lipoproteins of Sf 20-5000 from fat-fed normal subjects inhibited the degradation of 125I-labeled rabbit beta-VLDL as effectively as nonradioactive rabbit beta-VLDL. We conclude: 1) the beta-VLDL receptor is genetically distinct from the LDL receptor, and 2) intestinally derived human lipoproteins are recognized by the beta-VLDL receptor on macrophages.

Antibody against low density lipoprotein receptor blocks uptake of low density lipoprotein (but not high density lipoprotein) by the adrenal gland of the mouse in vivo.

The adrenal gland of the mouse takes up intravenously administered 125I-labeled human low density lipoprotein (LDL) by a high affinity, receptor-mediated mechanism. Uptake is enhanced by treatment of mice with a combination of 4-aminopyrazolopyrimidine, which eliminates endogenous mouse lipoproteins from the plasma, and adrenocorticotropin, which increases the number of adrenal LDL receptors. In the current studies, we show that adrenal uptake of 125I-LDL is blocked when the mice have received a prior intravenous injection of a rabbit antibody directed against the LDL receptor purified from bovine adrenal cortex. The antibody-mediated inhibition of 125I-LDL uptake persisted for 6 h and was reversed by 19 h. Adrenal uptake of 125I-labeled high density lipoprotein was not affected by the antibody, supporting the previous suggestion that high density lipoprotein uptake by the adrenal gland is mediated by a receptor that differs from the LDL receptor. The current studies illustrate the usefulness of antibodies in probing the process of receptor-mediated endocytosis in intact animals.

LDL receptors in coated vesicles isolated from bovine adrenal cortex: Binding sites unmasked by detergent treatment

Coated vesicles isolated from bovine adrenal cortex contain specific binding sites that recognize 125I-labeled human low density lipoprotein (LDL). These sites share the properties of the functional LDL receptors previously demonstrated on the surface of adrenal cells and in unfractionated adrenal membranes. Approximately 90% of the LDL receptors of the isolated coated vesicles were initially masked. Binding of 125I-LDL increased 10 fold after the vesicles were disrupted with the detergent octylglucoside. The LDL receptors of intact coated vesicles were also shielded from destruction by pronase; proteolytic destruction occurred only after the vesicles had been disrupted with octylglucoside. The adrenal coated vesicles measured 60 nm in diameter, suggesting that they were derived from the Golgi apparatus. Like the previously studied coated vesicles from brain and other tissues, the coated vesicles from adrenal cortex contained clathrin as the major protein component. In contrast to the coated vesicles of adrenal cortex, however, the brain coated vesicles failed to reveal masked LDL receptor activity when treated with octylglucoside. The current date indicate that isolated coated vesicles from the adrenal cortex contain LDL receptors and that these receptors exist in a masked form, apparently because their binding sites face the interior of the vesicle.

Regulation of low density lipoprotein receptors by adrenocorticotropin in the adrenal gland of mice and rats in vivo.

Membranes prepared from the adrenal gland of mice and rats possess high affinity binding sites that recognize 125I-labeled human low density lipoprotein (LDL). These binding sites resemble the functional LDL receptors that mediate the uptake of LDL by cultured mouse and bovine adrenal cells. The number of LDL binding sites per mg of membrane protein increased 2- to 5-fold over 24 h when mice or rats were treated with adrenocorticotropin (ACTH). In rats, this increase was accompanied by a similar ACTH-induced increase in the adrenal uptake of intravenously administered 125I-LDL, suggesting that the LDL binding sites mediate the uptake of LDL by the adrenal in the intact animal. The number of LDL binding sites on adrenal membranes rose by 5-fold when animals were rendered lipoprotein-deficient, either by treatment of mice with 4-aminopyrazolopyrimidine or by treatment of rats with 17 alpha-ethinyl estradiol. This increase was prevented when endogenous ACTH secretion was blocked by administration of dexamethasone, suggesting that ACTH was required. The current experiments suggest that LDL receptors provide one source of cholesterol for the mouse and rat adrenal in vivo and that the number of LDL receptors of this organ is regulated by ACTH.