Rat adrenal uptake and metabolism of high density lipoprotein cholesteryl ester

Metabolism of high density lipoprotein (HDL) cholesteryl ester (CE) by cultured rat adrenal cells was studied. Addition of [3H]CE-HDL to cells pretreated with adrenocorticotrophin in lipoprotein poor media resulted in a time- and concentration-dependent accumulation of [3H]cholesteryl ester and production of [3H]cholesterol and [3H]corticosterone. HDL-CE metabolism could be described as the sum of a high affinity ([ HDL-cholesterol]1/2 max = 16 micrograms/ml) and low affinity ([ HDL-cholesterol]1/2 max greater than 70 micrograms/ml) process. [3H]Cholesterol was found both intracellularly and in the media. Accumulation of [3H]cholesteryl ester could not be attributed to uptake and re-esterification of unesterified cholesterol since addition of Sandoz 58-035, an inhibitor of acyl coenzyme A:cholesterol acyltransferase, did not prevent ester accumulation. Moreover, addition of chloroquine did not inhibit cholesteryl ester hydrolysis indicating that hydrolysis was not lysosomally mediated. Aminoglutethimide prevented conversion of [3H]CE-HDL to steroid hormones but did not inhibit [3H]cholesteryl ester uptake. Cellular accumulation of [3H] cholesteryl ester exceeded accumulation of 125I-apoproteins 5-fold at 1 h and 35-fold at 24 h indicating selective uptake of cholesteryl ester moiety. We conclude that rat adrenal cells possess a mechanism for selective uptake of HDL cholesteryl esters which provides substrate for steroidogenesis. These results constitute the first direct demonstration that cholesteryl esters in HDL can be used as steroidogenic substrate by the rat adrenal cortex.     

Reactivity of HDL subfractions towards lecithin-cholesterol acyltransferase. Modulation by their content in free cholesterol

(1) Human HDL2 (d 1.070-1.125) and HDL3 (d 1.125-1.21) labelled with unesterified [14C]cholesterol, were incubated with a source of lecithin-cholesterol acyltransferase. For optimal activity, the reaction required the addition of albumin in excess, at least 3-times greater than the concentration of HDL-free cholesterol. Under such conditions, the reaction appeared saturable. HDL3 was found the most efficient substrate and the Vmax values expressed for 1.5 IU LCAT/ml and with an albumin/free cholesterol ratio of 3, were 8.3 nmol free cholesterol esterified/ml per h and 4.1 nmol/ml per h for HDL3 and HDL2, respectively. (2) HDL3 were modified in the presence of VLDL by inducing triacylglycerol lipolysis with a semipurified lipoprotein lipase from bovine milk. The newly formed HDL had gained free cholesterol and phospholipids, so that about 50% of these modified HDL, referred to as light-LIP-HDL3, were reisolated in the HDL2 density range. Light-LIP-HDL3 were enriched mostly in free cholesterol (+ 160%) and in phospholipid (+ 40%). Their reactivity towards LCAT was half-reduced compared to parent HDL3, which correlated well with a decrease in their phospholipid/free cholesterol molar ratio. Moreover, HDL3 artificially enriched in free cholesterol and exhibiting a comparable PL/FC behaved like lipolysis-modified HDL in their reactivity towards LCAT. (3) HDL3 were also modified by co-incubation with VLDL (post-VLDL-HDL3), or with VLDL and a source of lipid transfer protein (CET-HDL3). The latter treatment greatly affected the lipid composition of the core particle (-25% esterified cholesterol, +190% TG). In both cases, the moderate decreasing LCAT reactivity observed could be related to the phospholipid/free cholesterol ratio. Thus, like in artificial substrates, the lipid composition of the HDL surface may control the rate of LCAT-mediated cholesterol esterification.     

Factors influencing interaction of human plasma low-density lipoproteins with discoidal complexes of apolipoprotein A-I and phosphatidylcholine

The effect of plasma components on the particle size distribution and chemical composition of human plasma low-density lipoproteins (LDL) during interaction with discoidal complexes of human apolipoprotein A-I and phosphatidylcholine (PC) was investigated. Incubation (37 degrees C, 1 h and 6 h) of LDL with discoidal complexes in the presence of the plasma ultracentrifugal d greater than 1.20 g/ml fraction (activity of lecithin-cholesterol acyltransferase inhibited) produces an increase in LDL apparent particle diameter two-to six-fold greater than that observed in the absence of the plasma d greater than 1.20 g/ml fraction. In incubation mixtures of LDL and discoidal complexes, both in the presence and absence of the plasma d greater than 1.20 g/ml fraction, the extent of LDL apparent particle diameter increase is: (1) approximately three-fold greater at 6 h than at 1 h, and (2) markedly greater for LDL with initially small (22.4-24.0 nm) major components than for LDL with initially large (26.2-26.8 nm) major components. The facilitation factor in the plasma d greater than 1.20 g/ml fraction is not plasma phospholipid transfer protein. Purified human serum albumin produces an apparent particle diameter increase comparable to the plasma d greater than 1.20 g/ml fraction. The discoidal complex-induced increase in LDL apparent particle diameter value by albumin is associated with an increase in phospholipid uptake by LDL and a decreased loss of LDL unesterified cholesterol. In preliminary experiments, high-density lipoproteins (HDL) reverse the apparent particle diameter increase originally induced by discoidal complexes. The presence of HDL (HDL phospholipid/LDL phospholipid molar ratio of 10:1) in the incubation (6 h) mixture of LDL and discoidal complexes also attenuates LDL apparent particle diameter increase. In vivo, the plasma LDL/HDL ratio may be a controlling factor in determining the extent to which phospholipid uptake and the associated change in LDL particle size distribution occurs.     

Thermal behavior of human plasma high density lipoprotein.

Human plasma low density lipoprotein displays a reversible thermal transition between 20 and 40 degrees C, due to a phase transition of its core cholesterol ester from a smectic to a more liquid-like state. To determine if the cholesterol of high density lipoprotein (HDL) displays similar thermal behavior, the human lipoprotein and its extracted lipid have been examined by differential scanning calorimetry, low angle X-ray scattering and polarizing microscopy. Neither HDL2**(d 1.063--1.125--1.21 g/ml) nor HDL3(d1.125--1.21g/ml) show thermal transitions between O and 60 degrees C. By contrast cholesterol ester isolated from HDL and mixtures of cholesterol oleate and linoleate show reversible liquid crystalline transitions between 20 and 40 degreesC. X-ray scattering studies of HDL2 and HDL3 performed at 10 degreesC show no scattering fringes attributable to a smectic phase of cholesterol ester. When HDL is heated to temperatures above 60 degreesC a broad, double-peaked endotherm is observed. The first component (peak temperature=71 degreesC) corresponds to a selective release of apoprotein A-1 from the lipoprotein, and the second component (peak temperature=90 degreesC) to a more generalized disruption of lipoprotein structure with release of cholesterol ester and apoprotein A-2. Following the thermal disruption of HDL, reversible liquid crystalline transitions of cholesterol ester can be seen by differential scanning calorimetry and polarizing microscopy, showing the presence of large domains of cholesterol ester. The absence of cholesterol ester transitions in intact HDL may indicate an interaction of cholesterol ester molecules with the protein-phospholipid surface of HDL that prevents the formation of an organized lipid phase. The high temperature behavior of HDL indicates that apoprotein A-1 is less important than apoprotein A-2 in maintaining the HDL apolar lipids in the form of a stable miroemulsion.     

Lecithin:cholesterol acyltransferase-induced transformation of HepG2 lipoproteins

Previous studies with the human hepatoblastoma-derived HepG2 cell line in this laboratory have shown that these cells produce high density lipoproteins (HDL) that are similar to HDL isolated from patients with familial lecithin:cholesterol acyltransferase (LCAT) deficiency. Experiments were, therefore, performed to determine whether HepG2 HDL could be transformed into plasma-like particles by incubation with LCAT. Concentrated HepG2 lipoproteins (d less than 1.235 g/ml) were incubated with purified LCAT or lipoprotein-deficient plasma (LPDP) for 4, 12, or 24 h at 37 degrees C. HDL isolated from control samples possessed excess phospholipid and unesterified cholesterol relative to plasma HDL and appeared as a mixed population of small spherical (7.8 +/- 1.3 nm) and larger discoidal particles (17.7 +/- 4.9 nm long axis) by electron microscopy. Nondenaturing gradient gel analysis (GGE) of control HDL showed major peaks banding at 7.4, 10.0, 11.1, 12.2, and 14.7 nm. Following 4-h LCAT and 12-h LPDP incubations, HepG2 HDL were mostly spherical by electron microscopy and showed major peaks at 10.1 and 8.1 nm (LCAT) and 10.0 and 8.4 nm (LPDP) by GGE; the particle size distribution was similar to that of plasma HDL. In addition, the chemical composition of HepG2 HDL at these incubation times approximated that of plasma HDL. Molar increases in HDL cholesteryl ester were accompanied by equimolar decreases in phospholipid and unesterified cholesterol. HepG2 low density lipoproteins (LDL) isolated from control samples showed a prominent protein band at 25.6 nm with GGE. Active LPDP or LCAT incubations resulted in the appearance of additional protein bands at 24.6 and 24.1 nm. No morphological changes were observed with electron microscopy. Chemical analysis indicated that the LDL cholesteryl ester formed was insufficient to account for phospholipid lost, suggesting that LCAT phospholipase activity occurred without concomitant cholesterol esterification.     

Determination of lecithin:cholesterol acyltransfer in mouse plasma and the influence of mercaptoethanol and sulphydryl blocking agents on its activity.

1. The cholesterol esterifying activity in mouse plasma has been identified as lecithin:cholesterol acyltransferase (LCAT) on the basis of stoichiometric data, predominant transfer of polyunsaturated fatty acids, wide pH optimum and inhibition of esterification by phospholipase A2 and sulphydryl blocking agents. The esterifying activity differed from that present in plasma of man, rat and other species since it was partially inhibited by mercaptoethanol and other thiols. 2. Stoichiometric correlations between unesterified cholesterol, lecithin and lysolecithin were not exact, suggesting possible involvement of other enzymes in the overall esterification process during in vitro incubation of mouse plasma. 3. The initial rate of cholesterol esterification was determined by in vitro incubation of mouse plasma, whose cholesterol had been labelled by prior in vivo injection of 3H-mevalonic acid. The mean rate was 281 +/- 74 nmol/ml/hr (mean +/- S.D., n = 12) and correlated with unesterified cholesterol concentration (r = 0.73, P less than 0.01).     

A 70-kDa apolipoprotein designated ApoJ is a marker for subclasses of human plasma high density lipoproteins

A new apolipoprotein, termed apolipoprotein J (apoJ), was purified from human plasma by immunoaffinity chromatography. ApoJ is a glycoprotein consisting of disulfide-linked subunits of 34-36 and 36-39 kDa. Each subunit is glycosylated and has a pI range of 4.9-5.4. ApoJ exists in the plasma associated with high density lipoproteins (HDL) and specifically with subclasses of HDL which also contain apoAI and cholesteryl ester transfer protein activity. Immunoaffinity purified apoJ-HDL subclasses have apparent molecular masses of 80, 160, 240, 340, and 520 kDa, as determined by gradient gel electrophoresis. By negative staining electron microscopy, apoJ-HDL range in diameter from 5 to 16 nm. Fractionation of plasma by vertical gradient density centrifugation revealed apoJ-HDL in HDL2 (d 1.063-1.125 g/ml) with the majority overlapping HDL3 (d 1.125-1.21 g/ml) and very high density lipoprotein (d 1.21-1.25 g/ml). The bimodal density distribution of apoJ-HDL suggests that these subclasses have a unique metabolic relationship and may play a role in the transport of cholesterol from peripheral tissues to the liver.     

Characterization of lipoproteins produced by the human liver cell line, Hep G2, under defined conditions

Confluent monolayers of the human hepatoblastoma-derived cell line, Hep G2, were incubated in serum-free medium. Conditioned medium was ultracentrifugally separated into d less than 1.063 g/ml and d 1.063-1.20 g/ml fractions since very little VLDL was observed. The d less than 1.063 g/ml fraction was examined by electron microscopy; it contained particles of 24.5 +/- 2.3 nm diameter, similar in size to plasma LDL; a similar size was demonstrated by nondenaturing gradient gel electrophoresis. These particles possessed apoB-100 only. The d less than 1.063 g/ml fraction had a lipid composition unlike that of plasma LDL; unesterified cholesterol was elevated, there was relatively little cholesteryl ester, and triglyceride was the major core lipid. The d 1.063-1.20 g/ml fraction was heterogeneous in size and morphology. Electron microscopy revealed discoidal particles (14.9 +/- 3.2 nm long axis and 4.5 +/- 0.2 nm short axis) as well as small spherical ones (7.6 +/- 1.4 nm diameter). Nondenaturing gradient gel electrophoresis consistently showed the presence of peaks at 13.4 11.9, 9.7, and 7.4 nm. The latter peak was conspicuous and probably corresponded to the small spherical structures seen by electron microscopy. Unlike plasma HDL, Hep G2 d 1.063-1.20 g/ml lipoproteins contained little or no stainable material in the (HDL3a)gge region by gradient gel electrophoresis. Hep G2 d 1.063-1.20 g/ml lipoproteins differed significantly in composition from their plasma counterparts; unesterified cholesterol and phospholipid were elevated and the mole ratio of unesterified cholesterol to phospholipid was 0.8. Cholesteryl ester content was extremely low. ApoA-I was the major apolipoprotein, while apoE was the next most abundant protein; small quantities of apoA-II and apoCs were also present. Immunoblot analysis of the d 1.063-1.20 g/ml fraction after gradient gel electrophoresis showed that apoE was localized in the larger pore region of the gel (apparent diameter greater than 12.2 nm); the apoA-I distribution in this fraction was very broad (7.1-12.2 nm), and included a distinct band at 7.4 nm. Immunoblotting after gradient gel electrophoresis of concentrated medium revealed that a significant fraction of apoA-I in the uncentrifuged medium was in a lipid-poor or lipid-free form. This cell line may be a useful model for investigating the metabolism of newly formed HDL.     

Characterization of high density lipoprotein binding activity in rat adrenocortical cells

Rat adrenocortical cells take up high density lipoprotein cholesterol for use as steroidogenic substrate. To better understand this unique uptake process, we have first characterized HDL binding. Infusion of human 125I-labeled HDL into rats pretreated with 4-APP demonstrated that the adrenal and ovary accumulate HDL in a saturable fashion in vivo. Subsequent studies using isolated rat adrenocortical cells demonstrated that cellular uptake of HDL is comprised of two events. One event is characterized by reversible membrane binding and is complete by 60 min (t1/2 = 20 min). The second event is marked by irreversible apoprotein accumulation which continues for at least 3 hr. Reversibly bound material exhibits the same apoprotein distribution as unincubated HDL. Irreversible accumulation could not be attributed to internalization or lysosomal accumulation inasmuch as it also occurred with partially purified plasma membranes and was not enhanced by addition of chloroquine. Reversible binding of human HDL3 exhibited a saturable dependence on concentration (Kd = 27 micrograms protein/ml; N = 3.0 X 10(6) sites/cell) similar to that previously reported for rat liver, ovary, and testis. Cell accumulation of HDL decreased by over 80% at 4 degrees C compared to 37 degrees C, did not require calcium, and was not diminished by prior cell treatment with trypsin or pronase. These results indicate that rat adrenocortical cells possess plasma membrane recognition sites for HDL with different properties than those of the LDL receptor. Moreover, adrenal accumulation of HDL apoproteins does not lead to secondary lysosome formation.     

Phospholipase A2-treated human high-density lipoprotein and cholesterol movements: exchange processes and lecithin: cholesterol acyltransferase reactivity

Human HDL3 (d 1.125-1.21 g/ml) were treated by an exogenous phospholipase A2 from Crotalus adamenteus in the presence of albumin. Phosphatidylcholine hydrolysis ranged between 30 and 90% and the reisolated particle was essentially devoid of lipolysis products. (1) An exchange of free cholesterol was recorded between radiolabelled erythrocytes at 5-10% haematocrit and HDL3 (0.6 mM total cholesterol) from 0 to 12-15 h. Isotopic equilibration was reached. Kinetic analysis of the data indicated a constant rate of free cholesterol exchange of 13.0 microM/h with a half-time of equilibration around 3 h. Very similar values of cholesterol exchange, specific radioactivities and kinetic parameters were measured when phospholipase-treated HDL replaced control HDL. (2) The lecithin: cholesterol acyltransferase reactivity of HDL3, containing different amounts of phosphatidylcholine, as achieved by various degrees of phospholipase A2 treatment, was measured using a crude preparation of lecithin: cholesterol acyltransferase (the d 1.21-1.25 g/ml plasma fraction). The rate of esterification was determined between 0 and 12 h. Following a 15-30% lipolysis, the lecithin: cholesterol acyltransferase reactivity of HDL3 was reduced about 30-40%, and then continued to decrease, though more slowly, as the phospholipid content was further lowered in the particle. (3) The addition of the lecithin: cholesterol acyltransferase preparation into an incubation medium made of labelled erythrocytes and HDL3 promoted a movement of radioactive cholesterol out of cells, above the values of exchange, and an accumulation of cholesteryl esters in HDL. This reflected a mass consumption of free cholesterol, from both the cellular and the lipoprotein compartments upon the lecithin: cholesterol acyltransferase action. As a consequence of a decreased reactivity, phospholipase-treated HDL (with 2/3 of phosphatidylcholine hydrolyzed) proved much less effective in the lecithin: cholesterol acyltransferase-induced removal of cellular cholesterol.     

Induction of corticosteroid biosynthetic pathway by ACTH and high-density lipoprotein in newborn rat adrenocortical cells cultured in serum-free medium

In order to investigate the role of rat high-density lipoprotein (HDL) on adrenal cholesterol accumulation and steroidogenic pathways (corticosteroid, i.e., 21-hydroxysteroid biosynthesis and reductive metabolism of progesterone), newborn rat adrenal cells cultured in serum-free medium were used. Incubation of [4-14C]cholesterol-HDL in serum-free medium compared to those in medium with lipoprotein-deficient serum, in serum-free medium with ACTH compared to those without ACTH, both showed an increase of labelled cholesterol in cells and of labelled 21-hydroxysteroids excreted in medium. Substitution of serum-supplemented medium by serum-free and cholesterol-free medium led to a deep decrease of ACTH-induced steroid biosynthesis with a predominance of 20 alpha-reduced steroids; addition of HDL restored the corticosteroid biosynthesis and decreased the reductive metabolism. Addition of increased concentrations of HDL (7-150 micrograms cholesterol/ml) enhanced, in a saturable fashion, the total cholesterol uptake and the corticosteroid biosynthesis. The total cholesterol accumulation in cells exceeded by 4-fold the steroid production at saturation. The ratio between the two steroidogenic pathways increased up to 40 at saturation in favor of corticosteroids. These results suggest that HDL is at least partly internalized and that probably its constituents contribute greatly to the control of the two different steroidogenic pathways.     

Reverse cholesterol transport in the isolated perfused rat spleen.

1. A method has been developed which enables the rat spleen to be loaded in vivo with [3H]cholesterol to a high specific radioactivity using cholesterol-labelled erythrocytes. The erythrocytes were shown to be rapidly degraded by the spleen and not released intact during subsequent perfusion. 2. When labelled spleens were perfused with whole blood or serum, lipoproteins in the high-density lipoprotein (HDL) range were shown to be the principal lipoprotein vehicles for the removal of cholesterol, the specific radioactivity of cholesterol being much greater in the HDL fractions than in other lipoproteins, particularly in the d 1.175-1.210 fraction. 3. The formation of [3H]cholesteryl ester was restricted to the major HDL fractions. 4. Experiments utilizing individual HDL fractions added to a basal perfusate indicated that HDL1 (d 1.050-1.085) was of less importance in the removal of cholesterol from the spleen than HDL subfractions of higher density. Also, a decrease in density of the lipoproteins was observed during perfusion, concurrent with uptake of cholesterol, especially in the d 1.085-1.125 subfraction. 5. When [3H]cholesterol-labelled spleens were perfused with whole blood, about half of the radioactivity released was detected in erythrocytes, indicating a rapid exchange or transport of cholesterol. Thus erythrocytes could play an important role in the transfer of unesterified cholesterol when the chemical potential gradient is favourable.     

Conversion of human plasma high density lipoprotein-2 to high density lipoprotein-3. Roles of neutral lipid exchange and triglyceride lipases

Cholesterol esters accumulating in human plasma high density lipoproteins (HDL) are important in conversion of HDL3 to larger HDL2. We studied whether mechanisms of removal of cholesterol esters from HDL might be important in a reverse direction, i.e. conversion of HDL2 to HDL3. Native HDL2 or HDL3 is incubated with very low density lipoproteins (VLDL) and lipoprotein-poor plasma (d greater than 1.21 g/ml) at 37 degrees C. After incubation, "modified" (M) VLDL, and HDL2 or HDL3 are reisolated by ultracentrifugation. In modified M-HDL2 or M-HDL3, triglyceride becomes the major core lipid as the triglyceride/cholesterol ester weight ratio increases 8-10-fold relative to native HDL. With only small changes in protein/phospholipid ratios in M-HDLs, the large decrease in cholesterol ester/protein ratios suggest net cholesterol ester loss from HDL. Quantitative recovery analyses prove that the cholesterol esters lost from HDL are transferred to M-VLDL, which is now richer in cholesterol ester and poorer in triglyceride. These substantial exchanges of HDL lipids are not associated by significant transfer of HDL apoproteins but are dependent on neutral lipid transfer factors present in human lipoprotein-poor plasma (d greater than 1.21 g/ml). Similar results are obtained when purified core lipid transfer protein replaces d greater than 1.21 g/ml plasma in these incubations. After depletion of cholesterol ester from HDL, most but not all, exchanged triglyceride can be removed by lipolysis with either hepatic or lipoprotein lipase, resulting in a post-lipolysis HDL2 with an increased triglyceride content relative to normal HDL. With successive incubations with VLDL, and core lipid transfer factors, HDL2 loses more than two-thirds of its cholesterol esters. After lipolysis of acquired triglyceride, HDL2 is remodeled, in both composition and flotation parameters, toward HDL3.     

The alteration of membrane proteins in human erythrocyte membranes induced by quinolinic acid, an endogenous neurotoxin. Correlation of effect with structure

Mixtures containing subfractions of human plasma high-density lipoproteins (HDL) and human lipoprotein-free plasma were incubated in vitro at 37 degrees C. Esterification of cholesterol was observed both in incubations containing HDL-subfraction 3 (HDL3) and in those containing HDL-subfraction 2 (HDL2). The implication that the lecithin: cholesterol acyltransferase in lipoprotein-free plasma may therefore interact with lipoproteins in both HDL subfractions was developed further by proposing a simple model in which the two HDL subfractions may compete for interactions with the enzyme. This model was described mathematically and tested in experiments in which a constant amount of the enzyme was incubated with a wide range of concentrations of HDL2 and HDL3 present either alone or in combination. The model was able to predict experimentally observed rates of cholesterol esterification with great accuracy. The best fit was obtained with a Vmax for HDL3 that was 2.4-4-times greater than that for HDL2 and values of the apparent Km for HDL3 free cholesterol and HDL2 free cholesterol of 43-60 nmol/ml and 167-391 nmol/ml, respectively. The model thus predicts that, at physiological concentrations of lipoproteins, HDL2 will function as a competitive inhibitor of the cholesterol esterification reaction by displacing lecithin: cholesterol acyltransferase from a more effective substrate, HDL3, to a less effective substrate, HDL2.     

Relation of angiographically defined coronary artery disease to plasma lipoprotein subfractions and apolipoproteins.

The relation of coronary artery disease to plasma lipoproteins was examined in 104 men aged 35-65 years undergoing coronary angiography for suspected myocardial ischaemia. A score reflecting the number, degree, and length of stenoses in seven major coronary arteries was assigned to each angiogram. Lipid concentrations in lipoprotein subfractions were measured after preparative ultracentrifugation; plasma apolipoprotein concentrations were measured by electroimmunoassay. Men with high coronary scores tended to have lower plasma high-density lipoprotein (HDL) cholesterol concentrations and higher low-density lipoprotein (density 1.019-1.063 g/ml) cholesterol concentrations than subjects of similar age with low coronary scores (p approximately equal to 0.1). The strongest relation, however, was with the cholesterol concentration in the HDL2 subfraction (density 1.063-1.125 g/ml) of HDL, which averaged 44% lower in the severely affected patients (p less than 0.005). No associations were found between the coronary score and HDL3 cholesterol, the cholesterol content of lipoproteins of density less than 1.019 g/ml, plasma triglyceride, or the concentrations of apolipoproteins AI, AII, and E. The high coronary scores associated with low HDL2 concentrations reflected an increase in the number of both partial and complete stenoses distributed throughout the coronary tree. In contrast the sizes of the lesions and the proportion producing complete occlusion were unrelated to HDL2.     

Interaction of model discoidal complexes of phosphatidylcholine and apolipoprotein A-I with plasma components. Physical and chemical properties of the transformed complexes

Conversion of model discoidal complexes of egg yolk phosphatidylcholine and apolipoprotein A-I, upon interaction with a source of lecithin:cholesterol acyltransferase (plasma d greater than or equal to 1.21 g/ml fraction or partially purified enzyme) and with different sources of substrate unesterified cholesterol (LDL, VLDL or cholesterol incorporated into complexes), was investigated by gradient gel electrophoresis, gel filtration, equilibrium density gradient ultracentrifugation, electron microscopy and chemical analysis. When the incubation mixture contained an inhibitor of lecithin:cholesterol acyltransferase, discoidal complexes with mean long dimension of approximately 10.5 +/- 1.9 nm were converted (within 1 h) predominantly to small round particles and were partially depleted of their phospholipid content. Upon electrophoresis the small particles showed peak maxima within the migration intervals of the human plasma ( HDL3b ) gge and ( HDL3c ) gge subpopulations with associated particle size ranges of 7.8-8.2 and 7.2-7.8 nm, respectively. Within 1 h, in the presence of activated enzyme, the complexes were again converted in major part to the small particles. However, further incubation resulted in an apparent single-step conversion to a larger major product with peak maximum occurring within the migration intervals of the ( HDL2a ) gge and the ( HDL3a ) gge subpopulations (particle size ranges 8.8-9.8 and 8.2-8.8 nm, respectively). Formation of an apolar core was indicated by detection of cholesteryl esters in the conversion product. The form in which the substrate unesterified cholesterol was introduced did not markedly influence the size properties of the final conversion product. With VLDL as source of substrate, considerable incorporation of triacylglycerol occurred in company with a lower level of cholesteryl esters, suggesting transfer of these lipids during formation of the apolar core. Incubation of complexes with a partially purified (3000-fold) preparation of lecithin:cholesterol acyltransferase yielded a product similar in properties to that when the d greater than or equal to 1.21 g/ml fraction was used. Our model discoidal complexes and their conversion products exhibit properties very similar to those of potential precursors to HDL as well as of mature HDL particles. Their further investigation shows promise of providing detailed insight into the possible origin and heterogeneity of human plasma HDL.     

The effect of hypocholesterolemic drug treatment on adrenocortical cell function

The role of exogenous lipoprotein cholesterol versus endogenous cholesteryl esters as substrates in adrenal steroidogenesis was studied in isolated rat adrenal cells. Hypocholesterolemic drugs were used in rats to depress the plasma cholesterol concentration and the adrenal cholesterol concentration. Adrenal cortical cells were prepared in the usual way. The steroidogenic response to ACTH in normal adrenal cells and in cells which have been cholesterol-depleted was studied. Normal adrenal cells responded specifically over a 6 h incubation period to low doses of ACTH (half-maximal response equivalent to 40 microunits ACTH). These normal cells exhibited no altered response over a 3 h period to ACTH in the presence of serum or serum lipoproteins. The hypocholesterolemic drugs, 4-aminopyrazolo-[3,4-d]-pyrimidine, hexestrol and 17 alpha-ethinyl estradiol were used to lower plasma cholesterol, and after 1 day of 4-aminopyrazolo-[3,4-d]-pyrimidine and 5 days of hexestrol or 17 alpha-ethinyl estradiol treatment the plasma total cholesterol concentrations were similar. After 3 days of 4-aminopyrazolo-[3,4-d]-pyrimidine treatment the adrenal total cholesterol content was lower than after 1 day of this treatment, or 5 days of hexestrol treatment or 5 days of 17 alpha-ethinyl extradiol treatment. Lipoproteins had no significant effect on ACTH-stimulated steroidogenesis in cells isolated from rats treated for 1 day with 4-aminopyrazolo-[3,4-d]-pyrimidine, or for 5 days with hexestrol or 17 alpha-ethinyl estradiol. However, lipoproteins did stimulate steroidogenesis in cells from rats treated for 3 days with 4-aminopyrazolo-[3,4-d]-pyrimidine. The results show that normal adrenal cells contain a reserve of intracellular cholesterol so that the supply of endogenous cholesterol for steroidogenesis does not limit the response to ACTH and exogenous lipoproteins have no effect on steroidogenesis. However, if the cells are severely depleted of cholesterol then exogenous lipoproteins must be added for maximal steroidogenesis to occur.     

Surface exposure of apolipoproteins in high density lipoproteins. I. Reactivities with agarose-immobilized proteases.

The exposure of apolipoproteins at the surface of human plasma high density lipoproteins (HDL) was assessed by their accessibility to agarose-immobilized forms of trypsin and chymotrypsin. Proteolysis of lipid-free apolipoproteins and the lipoprotein subfractions HDL2 (d = 1.08--1.125 g/ml) and HDL3 (d = 1.125--1.195 g/ml) that differ in lipid-to-protein ratio was compared by polyacrylamide gel electrophoresis and isoelectric focusing of the apolipoproteins and peptide fragments and by quantitation of the various carboxyl-terminal groups formed. Gel filtration of the proteolyzed lipoproteins on Sephadex G-150 column indicated that more than 90% of the apolipoproteins and peptides remain associated with lipoprotein complexes. Proteolysis of lipoproteins occurred more slowly and with less fragmentation of the lipoproteins and apolipoproteins than proteolysis of thelipid-free apolipoproteins or the proteolysis of lipoproteins by soluble proteases reported by other investigators. The difference in lipid content of HDL2 and HDL3 made little difference in their proteolysis. Proteolysis of the lipoproteins by agarose-trypsin was more rapid at 37 degrees C than at 22 degrees C, but the proteolytic products were similar and differed from the products from the lipid free proteins. Peptide fragments from lipoproteins were larger than those from lipid-free proteins, which suggests masking of potentially cleavable groups by lipid. The amounts (mol/g protein) of new carboxyl-terminal tyrosine and phenylalanine released by agarose -chymotrypsin were much greater from the lipid-free proteins, but about 3/4 of the tryptophan residues were inacessible in both lipoproteins and lipid-free proteins. In agarose-trypsin digestion, lysine residues were slightly more masked than arginine in the absence of lipids and much more so in the lipoproteins. However, in the lipoproteins apoA-II, which contains lysine but no arginine, was cleaved more rapidly and extensively by agarose-trypsin than apoA-I.     

Mechanism of the HDL2 stimulation of progesterone secretion in cultured placental trophoblast

Lipoprotein cholesterol (C) supports the high rate of progesterone production by the human placenta as endogenous cholesterol synthesis is low. To study underlying mechanisms whereby lipoproteins, including high density lipoprotein-2 (HDL2), stimulate progesterone secretion, trophoblast cells were isolated from human term placentas and maintained in primary tissue culture. Lipoproteins were added at several concentrations and medium progesterone secretion was determined. HDL2 (d 1.063-1.125 g/ml) as well as low density lipoproteins (LDL) (d 1.019-1.063 g/ml) but not HDL3 (d 1.125-1.21 g/ml) stimulated progesterone secretion in a dose-dependent manner, with HDL2 cholesterol entering the cell and serving as substrate for progesterone synthesis. Conversely, LDL and HDL2 produced a significant decrease in [2-14C]acetate incorporation into cell cholesterol. Cholesterol-depleted lipoproteins did not stimulate progesterone secretion. The stimulating effect of LDL was abolished by apolipoprotein modification by cyclohexanedione or reductive methylation and by the addition of anti-LDL receptor antibody or 10 microM chloroquine to the medium. [14C]acetate conversion into cholesterol was accelerated by these procedures. However, HDL2 stimulation of progesterone secretion and reduction of [14C]acetate incorporation into cholesterol was not blocked by chemical modification of apolipoproteins, anti-LDL receptor antibody, or chloroquine. Treatment of HDL2 with tetranitromethane or dimethylsuberimidate also did not block the stimulation of progesterone. To determine whether the capacity of HDL2 to deliver cholesterol to the trophoblast cells was restricted to subfractions differing in apoE content, HDL2 was chromatographed on heparin-Sepharose and three fractions (A, B, and C) were obtained. Fraction A was poorest in apoE and free cholesterol, fraction B contained the majority of cholesterol, and fraction C was the richest in apoE and free cholesterol. When added to trophoblast cells, fraction A stimulated little progesterone secretion, fraction B stimulated moderately, and fraction C did so greatly. Modification of these subfractions with cyclohexanedione or reductive methylation did not inhibit these effects. In conclusion, HDL2 stimulated progesterone secretion in human trophoblast cell culture. Contrary to LDL, the HDL effect was not mediated by apolipoproteins or the LDL receptor pathway. The ability of HDL2 to stimulate progesterone secretion is consistent with the passive transfer of free cholesterol to the cell membrane from a physicochemically specific subfraction of HDL. This mechanism may be an auxiliary source of cholesterol for human steroidogenic cells.     

Interactions between model triacylglycerol-rich lipoproteins and high-density lipoproteins in rat, rabbit and man

There are inverse relationships between HDL cholesterol and plasma triacylglycerol concentrations in normal and in hypertriglyceridemic individuals. To investigate the interactions between triacylglycerol-rich lipid particles and HDL, a lipid emulsion model of the triacylglycerol-rich lipoproteins was prepared. When emulsion particles were incubated with rat high-density lipoproteins (HDL) in the presence of lipid transfer activity (d greater than 1.21 g/ml fractions) from rabbit or human plasma there was a rapid bi-directional exchange of cholesteryl oleate (CO) and phospholipid (PL) labels between lighter and heavier fractions of HDL and emulsion particles. The transfers of CO and PL labels between both light and heavy fractions of HDL and the emulsion particles were increased with increasing amounts of emulsion added to the incubations. Incubation with the d greater than 1.21 g/ml fraction from rat plasma resulted in only a small exchange of CO whereas PL exchange was similar to rabbit and human plasma. Retinyl palmitate label was not transferred from emulsion particles to the HDL fractions even in the presence of lipid transfer activity from rabbit or human plasma. The present study shows that the transfer protein-mediated exchanges of surface and core lipids between HDL and the triacylglycerol-rich lipoproteins are affected by the quantity of triacylglycerol-rich particles in the system. This mechanism may contribute to the inverse relationships between plasma triacylglycerol concentrations and HDL concentrations in normal and hypertriglyceridemic individuals.