High Affinity Low Density Lipoprotein Research Articles

Dissociation of the High Density Lipoprotein and Low Density Lipoprotein Binding Activities of Murine Scavenger Receptor Class B Type I (mSR-BI) Using Retrovirus Library-based Activity Dissection

The murine class B, type I scavenger receptor (mSR-BI) is a receptor for both high density lipoprotein (HDL) and low density lipoprotein (LDL) and mediates selective, rather than endocytic, uptake of lipoprotein lipid. We have developed a "retrovirus library-based activity dissection" method to generate mSR-BI mutants in which some, but not all, of the activities of this multifunctional protein have been disrupted. This method employs three techniques: 1) efficient in vitro cDNA mutagenesis (here error-prone PCR was used), 2) efficient retroviral delivery and high expression of single mutant cDNAs into individual cells, and 3) isolation of infected cells expressing the desired mutant phenotype using high sensitivity positive/negative screening by two-color fluorescence-activated cell sorting. A set of mutants, all having arginine substitutions at two common sites (positions 402 or 401 and position 418), were isolated and characterized. Mutation at either site alone did not generate as strong a mutant phenotype (loss of DiI uptake from DiI-HDL) as did the double mutations. "Activity-dissected" double mutants were as effective as wild-type mSR-BI in functioning as LDL receptors, mediating high affinity LDL binding and uptake of metabolically active cholesterol from LDL, but they lost most of their corresponding HDL receptor activity. Thus, these mutants provide support for the proposal that the interaction of SR-BI with HDL differs from that with LDL. Examination of the in vivo function of such mutants may provide insights into the differential roles of the LDL and HDL receptor activities of SR-BI in normal lipoprotein metabolism and in SR-BI's ability to protect against atherosclerosis.

Platelet high affinity low density lipoprotein binding and import of lipoprotein derived phospholipids

The binding of low density lipoprotein (LDL) to the platelet cell membrane could facilitate the transfer of phospholipids from LDL to the platelets. A polyclonal antibody against the platelet glycoproteins IIb/IIIa inhibited the high affinity binding of 125I-LDL by up to 80%. The transfer of pyrene (py)-labeled sphingomyelin (SM), phosphatidylcholine and phosphatidylethanolamine from LDL to the platelets was unaffected by the antibody. The lectin wheat germ agglutinin (WGA) reduced the binding of 125I-LDL to the platelets by approximately 80%. In contrast, the lectin stimulated the transfer of SM from LDL into the platelets by about three-fold. WGA also specifically augmented the transfer of py-SM between lipid vesicles and the platelets, the stimulation being abolished in the presence of N-acetylglucosamine. Dextran sulfate (DS) increased the specific binding of 125I-LDL to the platelets by up to 2.8-fold. On the other hand, the import of LDL-derived py-phospholipids was unaffected by DS. Together, the results indicate that the phospholipid transfer from LDL to the platelets is independent of the high affinity LDL binding to the platelets and is specifically stimulated by WGA. Thus, the interactions of platelets with LDL phospholipids differ markedly from those with the apoprotein components of the lipoproteins.

Low Density Lipoprotein Binding and Uptake by Human and Rat Islet Cells

Abnormalities in lipoprotein metabolism are common in diabetes. It is unknown whether variations in form or concentration of lipoproteins influence the function of pancreatic β cells. This study investigates whether low density lipoproteins (LDL) exhibit specific interactions with islet β cells. Radioactively labeled LDL (125I-LDL) and fluorescently labeled LDL (DiI-LDL) were used as tracers. Rat islet cells express high affinity LDL binding sites (Kd = 9 nm), which are also recognized by very low density lipoproteins and which are down-regulated by LDL. Binding of LDL appears restricted to the β cells, as it was not detected on islet endocrine non-β cells. At 37 C, LDL is taken up and lysosomally degraded by islet β cells but not by islet non-β cells. Human islet cells were also found to present LDL binding, uptake, and degradation. Compared with rat islet cells, human islet cells exhibit 10-fold less binding sites (2.107vs. 2.108 per 103 cells) with a 2-fold lower Kd value (5 nm) and an equal sensitivity to LDL-induced down-regulation. In conclusion, human and rat islet β cells express LDL receptors that can internalize the lipoprotein. This pathway should be examined for its potential role in (dys)regulating pancreatic β cell functions.

Low density lipoprotein binding and uptake by human and rat islet beta cells.

Abnormalities in lipoprotein metabolism are common in diabetes. It is unknown whether variations in form or concentration of lipoproteins influence the function of pancreatic beta cells. This study investigates whether low density lipoproteins (LDL) exhibit specific interactions with islet beta cells. Radioactively labeled LDL (125I-LDL) and fluorescently labeled LDL (DiI-LDL) were used as tracers. Rat islet cells express high affinity LDL binding sites (K(d) = 9 nM), which are also recognized by very low density lipoproteins and which are down-regulated by LDL. Binding of LDL appears restricted to the beta cells, as it was not detected on islet endocrine non-beta cells. At 37 C, LDL is taken up and lysosomally degraded by islet beta cells but not by islet non-beta cells. Human islet cells were also found to present LDL binding, uptake, and degradation. Compared with rat islet cells, human islet cells exhibit 10-fold less binding sites (2.10(7) vs. 2.10(8) per 10(3) cells) with a 2-fold lower K(d) value (5 nM) and an equal sensitivity to LDL-induced down-regulation. In conclusion, human and rat islet beta cells express LDL receptors that can internalize the lipoprotein. This pathway should be examined for its potential role in (dys)regulating pancreatic beta cell functions.

Mechanisms of regulation of ovarian sterol metabolism by insulin-like growth factor type II: in vitro studies with swine granulosa cells.

The present studies were designed to investigate the nature of the actions of insulin-like growth factor-II (IGF-II) on granulosa cell steroidogenesis and assess the potential facilitative interactions between IGF-II and other major regulators of ovarian sterol metabolism, viz. estrogen, FSH, and low density lipoprotein (LDL). In serum-free first passage monolayer cultures of swine granulosa cells, human recombinant IGF-II stimulated progesterone production with a half-maximally effective concentration of 4.6 +/- 1.2 ng/ml (0.61 +/- 0.16 nM) between 0-48 h of culture and 27 +/- 5.7 ng/ml (3.6 +/- 0.76 nM) between 48-96 h. Maximal progesterone accumulation increased 12-fold over that in untreated cultures (48-96 h). Over the latter interval, IGF-I stimulated progesterone production approximately 10-fold, with a significantly lower ED50 of 6.1 +/- 0.70 ng/ml (0.78 +/- 0.09 nM; P < 0.01 vs. IGF-II effect). IGF-II (100 ng/ml) enhanced progesterone biosynthesis approximately 2-fold in the presence of 25-hydroxycholesterol, suggesting that IGF-II increases the effective activity of the mitochondrial cholesterol side-chain cleavage enzyme. IGF-II (100 ng/ml) augmented human LDL-promoted progesterone production approximately 18-fold between 0-48 h of culture and approximately 6-fold between 48-96 h. In addition, IGF-II showed time-dependent stimulatory effects on the rates of [125I]iodo-LDL internalization, and the amounts of cell-associated and degraded lipoprotein. IGF-II increased by approximately 10-fold the number of specific high affinity LDL receptors on granulosa cells, with no apparent change in their binding affinity, as assessed in equilibrium competition studies. Coadministration of IGF-II and FSH (100 ng/ml) or estradiol (E2; 1 microgram/ml) for 2 days increased progesterone production synergistically. Cotreatment with FSH or E2 for 4 days decreased the ED50 of IGF-II's stimulation of progesterone accumulation by 61% and 50%, respectively (P < 0.01). Synergistic interactions also existed between IGF-II and 8-bromo-cAMP, which indicates that IGF-II can act in part at cellular loci distal to cAMP generation. Northern blot analysis of total RNA isolated from granulosa cells treated with IGF-II (100 ng/ml), FSH (100 ng/ml), or IGF-II plus FSH for 2 days revealed 5-, 7-, or 8-fold increases, respectively, in the amount of cytochrome P450 cholesterol side-chain cleavage enzyme mRNA. The same treatments produced 6-fold increases in the level of LDL receptor mRNA, as determined by solution hybridization/RNase protection assays.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence for up-regulated low density lipoprotein receptor in human lung adenocarcinoma cell line A549

Human lung adenocarcinoma cell line A549 was studied with respect to the metabolism of human low density lipoprotein (LDL) and 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR) activity. After incubation in medium containing lipoprotein-deficient serum (LPDS) for 24 h, the A549 cell line expresses a single class of high affinity LDL binding sites ( K D at 37°C of 15.1 ± 0.7 nM and capacity of 118 ± 2.8 ng/mg cell protein) and an HMGR activity of 111.4 ± 7 pmol/min/mg cell protein. After binding, the LDLs were internalized and degraded by a common saturable process. The HMGR activity was higher in A549 cells than in fibroblasts but LDL affinity and binding capacity were similar in both cell types. However, in the presence of lipoproteins, A549 cells showed a two-fold higher binding capacity than fibroblasts. When the cells were deprived of cholesterol, the amount of LDLR sites increased but the extent of stimulation was lower in A549 than in fibroblasts cells (2.5-fold versus six-fold respectively). This increase was accompanied by a similar increase in the specific LDLR mRNA cellular levels (two-fold versus six-fold respectively). When cells were deprived of exogenous and endogenous cholesterol (biosynthesis blocked by compactin), the binding capacity and the LDLR mRNA levels were yet again increased in A549 cells but not in fibroblasts. Taken together these results suggest that the level of expression of the LDLR is up-regulated in A549 cells compared to fibroblasts.

Lipoprotein(a) mediates high affinity low density lipoprotein association to receptor negative fibroblasts.

Lipoprotein(a) (Lp(a)) is an acute phase protein with unknown function. Lp(a) binds to low density lipoprotein (LDL) receptors, as well as to plasminogen (Plg) receptors. Preincubation of normal human skin fibroblasts with Lp(a) or with apo(a) cause a severalfold increase of LDL binding. Plg and kringle-4 of Plg have no effect. LDL receptor-negative fibroblasts respond upon preincubation with apo(a) with high affinity binding of LDL with Kd values that are almost identical with those of LDL binding to the LDL receptor. Incubation of apo(a)-pretreated fibroblasts with anti-apo(a) completely abolishes the increment of LDL binding. The high affinity LDL binding to LDL receptor-negative fibroblasts could be dissociated by approximately 80 and 54% with 5 mg/ml proline and 30 mg/ml NaCl, respectively, but not with dextran sulfate. The Lp(a)- and apo(a)-triggered LDL binding to fibroblasts have no effect on LDL internalization. These findings may reflect a key function in the role as an acute phase protein and may be relevant to the high atherogeneicity of Lp(a).

The low density lipoprotein receptor in Xenopus laevis. I. Five domains that resemble the human receptor

All five functional domains of the low density lipoprotein (LDL) receptor were assembled in their modern form more than 350 million years ago, as revealed from the sequence of two cloned cDNAs from the frog Xenopus laevis. The two cDNAs appear to represent duplicated copies of the LDL receptor gene that arose when the entire genome of Xenopus duplicated approximately 30 million years ago. Both frog LDL receptors bound Xenopus LDL with high affinity and human LDL with lower affinity when expressed in monkey COS cells. The receptors also showed high affinity for rabbit beta-migrating very low density lipoprotein and canine apoE-HDLc, both of which contain apolipoprotein E. Each of the seven cysteine-rich repeats in the ligand binding domain of the Xenopus receptors resembles its counterpart in the human, indicating that these repeats had already acquired their independent structures by the time of amphibian development. The cytoplasmic tail of both Xenopus receptors is 86% identical to the human, including the FDNPVY sequence necessary for internalization in coated pits. The attainment of a fully developed receptor structure in Xenopus suggests that earlier forms of the receptor may exist in animals that are older than amphibians. An accompanying paper demonstrates that expression of both Xenopus receptor genes is controlled by a sterol regulatory element that closely resembles the human sequence (Mehta, K.D., Brown, M.S., Bilheimer, D.W., and Goldstein, J.L. (1991) J. Biol. Chem. 266, 10415-10419).

Thyrotropin modulates low density lipoprotein binding activity in FRTL-5 thyroid cells.

FRTL-5 cells possess high affinity low density lipoprotein (LDL) receptors which bind, internalize, and degrade LDL. When FRTL-5 cells are deprived of thyrotropin (TSH) the binding of LDL increases more than 2-fold. Upon addition of TSH, at a concentration of 1 x 10(-10) M or greater, LDL binding decreases rapidly and within 24 h reaches the level which is typical of FRTL-5 cells chronically stimulated by TSH. The data available suggest that TSH-dependent down-regulation of LDL receptor activity is exerted through a reduction of the number of active LDL receptors, with no change in affinity. It is unlikely that the synthesis of LDL receptors is impaired, since LDL receptor messenger RNA is not decreased by TSH. The effect of the hormone on LDL receptor activity can be mimicked by 8-Br-cAMP and is completely abolished by the protein synthesis inhibitor cycloheximide but not by actinomycin D. TSH regulation of LDL receptor activity is lost in v-ras Ki-transformed FRTL-5 cells (Ki Mol) which also have lost TSH dependence for adenylate cyclase activation and growth. However, 8-Br-cAMP decreases LDL binding in Ki Mol FRTL-5 cells. The reduced availability of LDL receptor in TSH-stimulated FRTL-5 cells may be related to the increased membrane fluidity (Beguinot, F., Beguinot, L., Tramontano, D., Duilio, C., Formisano, S., Bifulco, M., Ambesi-Impiombato, F. S., and Aloj, S. M. (1987) J. Biol. Chem. 262, 1575-1582) or may reflect increased degradation of LDL receptors. We propose that a lower cholesterol uptake is needed in an actively proliferating cell population, to increase the production of isoprenoids whether it be for cholesterol biosynthesis or for the synthesis of other compounds requiring isoprenoid precursors.

Comparative study of the incorporation of ellipticine-esters into low density lipoprotein (LDL) and selective cell uptake of drug-LDL complex via the LDL receptor pathway in vitro

Esters of elliptinium with stearic (ST-NME), palmitic (PAL-NME) or oleic (OL-NME) acids, a series of lipophilic derivatives of ellipticine, were synthetized, in order to evaluate their incorporation into Low Density Lipoprotein (LDL). Among the three derivatives, OL-NME shows the most potent incorporation (83 μg/mg protein LDL) compared to ST-NME (37 μg/mg protein LDL) and PAL-NME (58 μg/mg protein LDL). The size of OL-NME-LDL was determined by size distribution particles, showing their homogeneity compared to native LDL. When culture normal human fibroblasts were incubated with [ 125I]LDL incorporated drug, they bound to the LDL receptor with the same affinity as native LDL and were internalized and degraded intracellularly. The presence of excess native LDL inhibited the cellular uptake and degradation of [ 125I]drug-LDL. We have used [ 125I]acetyl-LDL as a probe for a binding site on macrophages that mediated the uptake and degradation of chemically altered or denatured LDL. Mouse peritoneal macrophages were shown to take up and degrade [ 125I]acetyl-LDL at rates that were greater than those for the uptake and degradation of native [ 125I]LDL and [ 125I]drug-LDL. The in vitro cytotoxic test on L1210 murine leukemic cells demonstrated that the complex was cytotoxic and was more effective than the free drug. This cytotoxic activity of the drug-LDL complex depends on the LDL high affinity receptor since the addition of native LDL reduces the killing power. In contrast, methylated LDL, which does not bind to the LDL receptor, has no effect on it. We conclude that it is possible to incorporate a large amount of cytotoxic drug into LDL without modifying their cellular metabolism via the high affinity LDL receptor pathway. It indicates also that the delivery of lipophilic drugs using LDL might provide distinct advantages over the use of synthetic carriers.

Unexpected failure of bile acid malabsorption to stimulate cholesterol synthesis in sitosterolemia with xanthomatosis. Comparison with lovastatin.

We examined the relationship between cholesterol synthesis and high affinity low density lipoprotein (LDL) catabolism in freshly isolated mononuclear leukocytes and plasma sterols and apolipoprotein concentrations in three homozygous and one heterozygous subject with sitosterolemia with xanthomatosis and in 12 control subjects. Observations in untreated subjects were compared during therapy with lovastatin or interruption of the enterohepatic circulation of bile acids. Plasma cholesterol, plant sterol, and apolipoprotein B concentrations declined more than 50% in the two homozygous sitosterolemic subjects after ileal bypass surgery. In contrast, plasma cholesterol, plant sterol, and apolipoprotein B concentrations remained constant in a homozygous sitosterolemic subject and declined only 7% in a heterozygous sitosterolemic subject during 20 weeks of lovastatin (40 mg/day) treatment compared to a 28% decrease in similarly treated control subjects. Lovastatin treatment decreased cholesterol synthesis more than 60% but did not increase high affinity catabolism of LDL further in the sitosterolemic cells, compared to a more than 20% rise in control mononuclear leukocytes. Conversely, bile acid malabsorption increased cholesterol synthesis 59%, total hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase activity 13%, and receptor-mediated LDL degradation 41% in control cells, but did not stimulate cholesterol synthesis or microsomal HMG-CoA reductase activity in sitosterolemic mononuclear leukocytes although receptor-mediated LDL catabolism rose an additional 26%. These results demonstrate a greater than expected decrease in plasma sterols and apolipoprotein B concentrations in sitosterolemic subjects after stimulation of bile acid synthesis because of the inability to up-regulate cholesterol production. We suggest that bile acid-sequestering drugs or ileal exclusion surgery may be more effective treatments to mobilize accumulated sterol deposits and prevent atherosclerosis in this disease.

Increased Expression of High-Affinity Low-Density Lipoprotein Receptors on Human T-Blasts

Like all cells, lymphocytes need cholesterol for proper function, a requirement met by a finely tuned homeostasis between intracellular synthesis and uptake from the environment via low-density lipoproteins (LDL). We used flow cytometry to analyze the receptor activity of resting cells and T blasts incubated/activated in serum-free culture medium, or in medium supplemented with 25-5,000 micrograms/ml LDL. Dioctadecyl-indocarbocyanine has proved to be a useful fluorescent probe for investigating the LDL receptor activity of lymphocytes. The results show the receptor activity of day-3 resting T cells to be reduced more than 50% by 50 microgram LDL/ml, whereas 100-fold higher concentrations are necessary to achieve the same level of reduction in day-3 PHA blasts. The LDL receptor activities of individual blood donors' resting T cells, in vitro cholesterol-deprived resting T cells, and activated T blasts, were compared using two analytical techniques: spectrofluorometric analysis of detergent-solubilized cell suspensions and flow cytometric analysis of single living cells. Receptor affinity was determined by Scatchard analysis of spectrofluorometric binding curves, and by Line-weaver-Burke plots of flow cytometric data. Both methods yielded essentially identical dissociation constants (Kd) for cholesterol-deprived resting T cells and mitogen-activated T blasts, which fell in the expected range for the high-affinity LDL receptor (4.1-8.9 nM). In addition, spectrofluorometric analysis, but not flow cytometry, permitted quantification of LDL uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Follicle-stimulating hormone regulates low density lipoprotein metabolism by swine granulosa cells.

FSH amplified the stimulatory effect of low density lipoprotein (LDL) on progesterone biosynthesis by primary cultures of swine granulosa cells in a synergistic manner. The mechanisms subserving this synergism included the following. 1) FSH increased by 2.1-fold the number of specific high affinity low density lipoprotein (LDL) receptors on granulosa cells, with no change in apparent binding affinity. 2) FSH augmented by 1.8- and 3.6-fold the maximal rates of [125I]iodo-LDL internalization and degradation, respectively, without altering half-maximally effective concentrations of LDL supporting these processes. 3) FSH increased significantly the mass (micrograms) of free and esterified cholesterol (measured by fluorometry) contained in granulosa cells. 4) FSH stimulated the intracellular accumulation of free [3H]cholesterol and [3H]cholesteryl ester from exogenous [3H]cholesteryl linoleate-labeled LDL and amplified [3H]progesterone secretion by granulosa cells exposed to this source of lipoprotein-borne sterol. 5) The stimulatory effects of FSH on LDL degradation and cholesterol side-chain cleavage activity were mimicked by other activators of granulosa cell cAMP production, e.g. forskolin and cholera toxin. We conclude that FSH and LDL synergistically enhance progesterone biosynthesis by swine granulosa cells via cellular mechanisms that result in significantly augmented binding, internalization, and degradation of LDL in concert with increased effectual utilization of sterol substrate in the cholesterol side-chain cleavage reaction. These observations are consistent with a key trophic role for FSH in regulating the sterol-metabolizing ability and steroidogenic differentiation of granulosa cells and indicate that such actions of FSH can be accounted for by activation of the cAMP-effector pathway.

Low-density lipoprotein endocytosis: I. Influence of the multivalent ligand cationized ferritin on normal and receptor-negative human fibroblasts

Based upon the observation that the multivalent ligand cationized ferritin (CF) alters the cell surface distribution of anionic domains and significantly enhances the adsorptive endocytosis of 125I-labeled human serum albumin, these studies were undertaken to probe the influence of CF on receptor-mediated low-density lipoprotein (LDL) endocytosis and the nature of the mechanisms involved. A brief 1-min exposure of normal receptor upregulated fibroblasts to CF (0.2 mg/ml) resulted in a significant decrease ( P < 0.001) in the subsequent internalization and degradation of 125I-LDL. Studies with receptor downregulated normal fibroblasts indicated that CF pretreatment did not measurably influence 125I-LDL internalization and only slightly inhibited its degradation ( P < 0.05). In contrast, CF pretreatment of FH receptor-negative mutant skin fibroblasts resulted in a modest but significant increase in both 125I-LDL internalization and degradation ( P < 0.05). Scatchard analyses of binding data indicated that CF-pretreated upregulated normal fibroblasts exhibit a single class of LDL binding sites with an affinity, K d = 24.7 ± 4.1 n M, almost 10-fold lower than the affinity of binding sites in untreated controls, K d = 3.2 ± 0.06 n M. Increasing either the concentration or the duration of CF exposure resulted in additional inhibition of LDL internalization and degradation associated primarily with a decrease in the number of LDL binding sites without any further change in binding affinity. Total cellular LDL receptor-mediated binding, measured using an octylglucoside solubilization-filtration assay, confirmed the CF-induced decrease in high-affinity LDL binding. Pulse-chase experiments showed that CF had no direct influence on LDL degradation, nor did it influence targeting of the LDL-containing endosome toward exocytosis. Further, restoration of LDL receptor function to control values after CF pretreatment required de novo protein synthesis. The normal feedback inhibition of HMG-CoA reductase activity was nearly abolished by CF pretreatment. Additionally, CF pretreatment was found to induce not only a redistribution of surface anionic sites, but also a very rapid internalization of surface components labeled with 4,4′-[ 3H]diisothiocyano-1,2-diphenylethane-2,2′-disulfonic acid. It is concluded that the inhibitory influence of CF on LDL endocytosis is mediated via a decrease in the affinity and in the number of functional LDL receptors.

The insulin-like growth factor, somatomedin-C, modulates low density lipoprotein metabolism by swine granulosa cells.

Insulin-like growth factor I (IGF-I) synergistically amplified the stimulatory effect of low density lipoprotein (LDL) on progesterone biosynthesis by primary cultures of swine ovarian cells. The mechanisms subserving this facilitative interaction included the following. IGF-I's synergism with LDL was associated with a decrease in the mean half-maximally stimulatory concentration of LDL from 20-3.5 micrograms/ml. IGF-I increased by 3- to 6-fold the number of specific high affinity LDL receptors on ovarian cells, with no change in apparent binding affinity. IGF-I augmented by 3- and 18-fold the maximal rates of [125I]iodo-LDL internalization and degradation, respectively, without altering half-maximally effective concentrations of LDL supporting these processes. IGF-I increased by 2- to 2.5-fold the total mass of free and esterified cholesterol contained in granulosa cells. IGF-I stimulated the intracellular accumulation of free [3H]cholesterol and [3H]cholesteryl ester from exogenous [3H]cholesteryl linoleate-labeled LDL, and amplified [3H]progesterone secretion by granulosa cells exposed to this source of lipoprotein-borne sterol. These actions of IGF-I were demonstrated at 30- to 100-fold lower concentrations of IGF-I than insulin. We conclude that IGF-I and LDL synergistically enhance progesterone biosynthesis by ovarian cells. This synergism occurs in part via mechanisms that regulate the effectual delivery of lipoprotein-borne cholesterol substrate into cellular sterol pools that participate in steroid hormone biosynthesis.

Accumulation of native and methylated low density lipoproteins by healing rabbit arterial wall.

To determine whether healing arterial wall accumulation of low density lipoproteins (LDL) is mediated by the high affinity LDL receptor, normocholesterolemic rabbits were injected with 125I-LDL, 99mTc-LDL, or the reductively methylated analogs of these compounds (125I-MeLDL, 99mTc-MeLDL), 1 month after balloon catheter deendothelialization of the abdominal aorta. If the mechanism of accumulation requires interaction with the LDL receptor, reductively methylated lipoproteins which do not bind to the receptor should not accumulate in healing arterial wall. Twenty-four hours after injection of labelled lipoproteins, each animal was injected with Evans blue dye, in order to distinguish reendothelialized from deendothelialized aorta. One hour after dye injection, the aorta was fixed, removed, divided into abdominal (ballooned) and thoracic (unballooned) regions and counted. For all lipoprotein preparations, there were three to four times as many counts in the abdominal as in the thoracic aorta. En face autoradiographs were made of the aortas that had been exposed to 125I-labelled lipoproteins. In the autoradiographs, the areas of the lowest activity corresponded to the centers of healing endothelial islands. The most intense radioactivity for both lipoproteins occurred in the region of the leading edge of the endothelial islands where active endothelial regeneration was in progress. The overall distribution of native and MeLDL accumulation was the same. The results suggest that low density lipoproteins are accumulated in areas of active endothelial regeneration by a mechanism that does not involve the high affinity LDL receptor.

Taurine enhances low density lipoprotein binding. Internalization and degradation by cultured Hep G2 cells.

To evaluate the impact of taurine on hepatic cholesterol catabolism low density lipoprotein (LDL) binding, internalization and degradation were measured in cultured Hep G2 cells. Preincubation of cells with 0.1-10 mM taurine for 24 h stimulated LDL receptor activity by as much as 100%. Only the high affinity LDL receptor activity (specific) was increased by taurine preincubation, whereas the low affinity receptor activity (nonspecific) remained unchanged. Scatchard analysis of the binding data revealed that taurine doubled the number of LDL receptors without affecting receptor affinity. Taurine-enhanced LDL receptor activity was most pronounced when LDL concentrations exceeded 100 micrograms/ml, but was noted at taurine concentrations as low as 0.1 mM (plasma level). Interestingly, taurine had no effect on LDL receptor activity when it was added simultaneously with 125I-LDL to Hep G2 cells, or when non-bile acid-producing human skin fibroblasts were tested. Stimulation of LDL receptor activity was also obtained with 10 mM cysteine, a taurine precursor, but not with glycine. Increased cellular concentrations of taurine and cysteine were associated with an elevated rate of bile acid synthesis and a reduced cellular free cholesterol concentration. The data suggest that taurine enhanced LDL receptor activity by sparing cysteine, a known sulfhydryl group donor and stimulator of 7 alpha-hydroxylase activity, and that the latter stimulated bile acid production leading to increased utilization of cellular free cholesterol and enhanced LDL uptake.

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.

Interaction of tryptic peptides of apolipoprotein B-100 with dimyristoylphosphatidylcholine

Apolipoprotein B-100, the major protein constituent of human plasma low-density lipoproteins (LDL), was carboxyamidomethylated, digested with trypsin and the water-soluble tryptic peptides were coincubated with liposomes of dimyristoylphosphatidylcholine (DMPC). At 24.3° C the peptides induced lipid solubilization as evidenced by optical clearing of the lipid-peptide mixture. Lipid-peptide complexes were isolated by density-gradient ultracentrifugation in KBr and had the following properties: DMPC/peptide ratio of 5.6 (w/w); buoyant density of 1.07–1.09 g/ml; discoidal morphology ( 51 ± 4 · 260 ± 28 A ̊ ) as determined by electron microscopy; and molecular weight of 1.5 · 10 6 as determined by nondenaturing polyacrylamide gel electrophoresis. Compared to liposomes and sonicated vesicles of DMPC, the lipid-peptide complexes had a more rigid structure as assessed by fluorescence polarization. Whereas intact LDL had 42% α-helix and 15% β-pleated sheet, the lipid-peptide complexes contained 70% α-helix and less than 5% β-pleated sheet. The lipid-peptide complexes did not bind to the fibroblast high-affinity LDL receptor. These results show that specific regions in apolipoprotein B-100 which interact with phospholipid have an amphipathic character and may represent primary sites for lipid-protein interaction in LDL.

The pathophysiology of cholesterol metabolism in man.

The concentration of low density lipoprotein in human plasma depends on the balance between its rates of synthesis and catabolism. Although both processes appear to be independently regulated they occur side by side in the liver and may be linked via the activity of the high affinity low density lipoprotein receptor on hepatocyte membranes. Dietary changes such as cholesterol feeding or variation in fat content can promote synthesis of the lipoprotein without changing catabolism while other interventions (e.g. sequestrant resin therapy) have the opposite effect. These different responses may be explained on the basis of compartmentalisation of regulatory sterol pools in the liver cell.