Protein heterogeneity of lipoprotein particles containing apolipoprotein A-I without apolipoprotein A-II and apolipoprotein A-I with apolipoprotein A-II isolated from human plasma

The protein heterogeneity of fractions isolated by immunoaffinity chromatography on anti-apolipoprotein A-I and anti-apolipoprotein A-II affinity columns was analyzed by high resolution two-dimensional gel electrophoresis. The two-dimensional gel electrophoresis profiles of the fractions were analyzed and automatically compared by the computer system MELANIE. Fractions containing apolipoproteins A-I + A-II and only A-I as the major protein components have been isolated from plasma and from high density lipoproteins prepared by ultracentrifugation. Similarities between the profiles of the fractions, as indicated by two-dimensional gel electrophoresis, suggested that those derived from plasma were equivalent to those from high density lipoproteins (HDL), which are particulate in nature. The established apolipoproteins (A-I, A-II, A-IV, C, D, and E) were visible and enriched in fractions from both plasma and HDL. However, plasma-derived fractions showed a much greater degree of protein heterogeneity due largely to enrichment in bands corresponding to six additional proteins. They were present in trace amounts in fractions isolated from HDL and certain of the proteins were visible in two-dimensional gel electrophoresis profiles of the plasma. These proteins are considered to be specifically associated with the immunoaffinity-isolated particles. They have been characterized in terms of Mr and pI. Computer-assisted measurements of protein spot-staining intensities suggest an asymmetric distribution of the proteins (as well as the established apolipoproteins), with four showing greater prominence in particles containing apolipoprotein A-I but no apolipoprotein A-II.     

The role of apolipoprotein A-I and apolipoprotein A-II in high-density lipoprotein binding to human adipocyte plasma membranes

Adipocyte plasma membranes purified from omental fat tissue biopsies of massively obese subjects possess specific binding sites for high-density lipoprotein (HDL3). This binding was independent of apolipoprotein E as HDL3 isolated from plasma of an apolipoprotein E-deficient individual was bound to a level comparable to that of normal HDL3. To examine the importance of apolipoprotein A-I, the major HDL3 apolipoprotein, in the specific binding of HDL3 to human adipocytes, HDL3 modified to contain varying proportions of apolipoproteins A-I and A-II was prepared by incubating normal HDL3 particles with different amounts of purified apolipoprotein A-II. As the apolipoproteins A-I-to-A-II ratio in HDL3 decreased, the binding of these particles to adipocyte plasma membranes was reduced. Compared to control HDL3, a 92 +/- 3.1% reduction (mean +/- S.E., n = 3) in maximum binding capacity was observed along with an increased binding affinity for HDL3 particles in which almost all of the apolipoprotein A-I had been replaced by A-II. The uptake of HDL cholesteryl ester by intact adipocytes as monitored by [3H]cholesteryl ether labeled HDL3, was also significantly reduced (about 35% reduction, P less than 0.005) by substituting apolipoprotein A-II for A-I in HDL3. These data suggest that HDL binding to human adipocyte membranes is mediated primarily by apolipoprotein A-I and that optimal delivery of cholesteryl ester from HDL to human adipocytes is also dependent on apolipoprotein A-I.     

Isolation and identification of HDL particles of low molecular weight

Small particles of high density lipoproteins (HDL) were isolated from fresh, fasting human plasma and from the ultracentrifugally isolated high density lipoprotein fraction by means of ultrafiltration through membranes of molecular weight cutoff of 70,000. These particles were found to contain cholesterol, phospholipids, and apolipoproteins A-I and A-II; moreover, they floated at a density of 1.21 kg/l. They contained 67.5% of their mass as protein and the rest as lipid. Two populations of small HDL particles were identified: one containing apolipoprotein A-I alone [(A-I)HDL] and the other containing both apolipoproteins A-I and A-II [A-I + A-II)HDL]. The molar ratio of apoA-I to apoA-II in the latter subclass isolated from plasma or HDL was 1:1. The molecular weights of these subpopulations were determined by nondenaturing gradient polyacrylamide gel electrophoresis and found to be 70,000; 1.5% of the plasma apoA-I was recovered in the plasma ultrafiltrate.     

Measurement of human high density lipoprotein apolipoprotein A-1 in serum by radioimmunoassay.

A sensitive and specific double antibody radioimmunoassay for the major apolipoprotein (apo A-I) of human serum high density lipoprotein (HDL) was developed. Initial studies indicated that direct measurements of apo A-I concentration in whole untreated sera or isolated high density lipoprotein fractions yielded variable results, which were lower than those obtained in the corresponding samples which had been subjected to delipidation. Subsequently, it was observed that heating diluted sera or HDL for 3 hr at 52 degrees C prior to assay resulted in maximal increases in apo A-I immunoreactivity to levels comparable to those found in the delipidated specimens. This simple procedure permitted multiple sera to be assayed efficiently with full recovery of apo A-I.     

Apolipoprotein A-II: beyond genetic associations with lipid disorders and insulin resistance

PURPOSE OF REVIEW: Apolipoprotein A-II, the second major HDL apolipoprotein, was often considered of minor importance relatively to apolipoprotein A-I and its role was controversial. This picture is now rapidly changing, due to novel polymorphisms and mutations, to the outcome of clinical trials, and to studies with transgenic mice. RECENT FINDINGS: The -265 T/C polymorphism supports a role for apolipoprotein A-II in postprandial very-low-density lipoprotein metabolism. Fibrates, which increase apolipoprotein A-II synthesis, significantly decrease the incidence of major coronary artery disease events, particularly in subjects with low HDL cholesterol, high plasma triglyceride, and high body weight. The comparison of transgenic mice overexpressing human or murine apolipoprotein A-II has highlighted major structural differences between the two proteins; they have opposite effects on HDL size, apolipoprotein A-I content, plasma concentration, and protection from oxidation. Human apolipoprotein A-II is more hydrophobic, displaces apolipoprotein A-I from HDL, accelerates apolipoprotein A-I catabolism, and its plasma concentration is decreased by fasting. Apolipoprotein A-II stimulates ATP binding cassette transporter 1-mediated cholesterol efflux. Human and murine apolipoprotein A-II differently affect glucose metabolism and insulin resistance. A novel beneficial role for apolipoprotein A-II in the pathogenesis of hepatitis C virus has been shown. SUMMARY: The hydrophobicity of human apolipoprotein A-II is a key regulatory factor of HDL metabolism. Due to the lower plasma apolipoprotein A-II concentration during fasting, measurements of apolipoprotein A-II in fed subjects are more relevant. More clinical studies are necessary to clarify the role of apolipoprotein A-II in well-characterized subsets of patients and in the insulin resistance syndrome.     

Identification and partial characterization of discrete apolipoprotein A-containing lipoprotein particles secreted by human hepatoma cell line HepG2

The purpose of this study was to identify the apolipoprotein A-containing lipoprotein particles produced by HepG2 cells. The apolipoprotein A-containing lipoproteins separated from apolipoprotein B-containing lipoproteins by affinity chromatography of culture medium on concanavalin A were fractionated on an immunosorber with monoclonal antibodies to apolipoprotein A-II. The retained fraction contained apolipoproteins A-I, A-II and E, while the unretained fraction contained apolipoproteins A-I and E. Both fractions were characterized by free cholesterol as the major and triglycerides and cholesterol esters as the minor neutral lipids. Further chromatography of both fractions on an immunosorber with monoclonal antibodies to apolipoprotein A-I showed that 1) apolipoprotein A-II only occurs in association with apolipoprotein A-I, 2) apolipoprotein A-IV is only present as part of a separate lipoprotein family (lipoprotein A-IV), and 3) apolipoprotein E-enriched lipoprotein A-I:A-II and lipoprotein A-I are the main apolipoprotein A-containing lipoproteins secreted by HepG2 cells.     

Isolation of apolipoproteins A-I, A-II and E and their estimation by rocket immunoelectrophoresis in blood plasma of dis-alpha-lipoproteinemic patients

The methods for isolation of pure apolipoproteins A-I, A-II and E from the blood plasma of donors for preparation of monospecific rabbit antisera against these apolipoproteins and their estimation in human blood plasma using immunoelectrophoresis are described. It was found that the average content of apolipoprotein A-I (apo A-I) in the blood plasma of healthy males is 126.6 mg%, that of apolipoprotein A-II (apo A-II) is 56.8 mg%, that of apolipoprotein E (apo E) is 10.2 mg%. The apo A-I content in blood plasma is increased in hyper-alpha-lipoproteinemic patients and is decreased in hypo-alpha-lipoproteinemic ones, i. e. there is a direct relationship between the changes in concentration of high density lipoproteins (HDL) and apo A-I. The concentration of apo A-II in dis-alpha-lipoproteinemias varies within a narrow range. A considerable increase of the alpha-cholesterol/apo A-I ratio suggesting an increased capacity of HDL to transport cholesterol in hyper-alpha-lipoproteinemic patients is observed. There exists an indirect correlation between the changes in the contents of apo A-I and apo E in dis-alpha-lipoproteinemic patients.     

Isolation and partial characterization of the lipoprotein families A and A-I from high-density lipoproteins of human serum

Procedures for the isolation of two lipoprotein fractions from plasma high-density lipoproteins (HDL), characterized by apolipoprotein A-I and apolipoprotein A-I together with apolipoprotein A-II, have been elaborated. Apolipoprotein A-I was identified as the protein moiety of one of these fractions (lipoprotein A-I) with polyacrylamide gel electrophoresis (at basic and acidic pH, as well as in the presence of sodium dodecyl sulphate), immuno-double-diffusion, and amino acid analysis. Apolipoproteins A-I and A-II were identified as the protein moiety of the other fraction (lipoprotein A) with polyacrylamide gel electrophoresis (basic and acidic pH) and immuno-double-diffusion. Lipoprotein A-I consisted of spherical particles with a diameter similar to that of HDL as judged from negative strains in the transmission electron microscope. The diameter was estimated to be 8.7 nm from gel chromatography. Lipoprotein A-I migrated in the HDL position on crossed immunoelectrophoresis. On iso-electric focusing lipoprotein A-I appeared as multiple bands in the pH range 5.05-5.55. Lipoprotein A-I had the density of an HDL-2 fraction (rho: 1.063-1.105). Lipoprotein A consisted of spherical particles with a diameter similar to that of HDL, as judged from negative strains in the transmission electron microscope. The diameter was estimated to be 7.9 nm from gel chromatography. The molar ratio between the A-I and A-II polypeptides was estimated to 1.3:1 with electroimmunoassay and calculations from the amino acid compositions. Lipoprotein A migrated in the position of HDL on crossed immuno-electrophoresis. On iso-electric focusing lipoprotein A appeared as one major and two minor bands in the pH range 5.10-5.30. Lipoprotein A had the hydrated density of an HDL-2 fraction.     

Lipid profile of body builders with and without self-administration of anabolic steroids

Twenty-four top-level body builders [13 anabolic steroid users (A); 11 non-users (N)] and 11 performance-matched controls (C) were examined to determine the effect on lipids, lipoproteins and apolipoproteins of many years of body building with and without simultaneous intake of anabolic steroids and testosterone. After an overnight fast, triglycerides (TG), total cholesterol (TOTC), high density lipoprotein cholesterol (HDLC), low density lipoprotein cholesterol (LDLC), the HDLC subfractions HDL2C and HDL3C, as well as apolipoprotein A-I (Apo A-I), apolipoprotein A-II (Apo A-II) and apolipoprotein B (Apo B) were determined. Both A and N, compared to C, showed significantly lower HDLC and higher LDLC concentrations, with the differences between A and C clearly pronounced. In a subgroup of 6 body builders taking anabolic steroids at the time of the study, HDLC, HDL2C, HDL3C, Apo A-I and Apo A-II were all significantly lower and LDLC was significantly higher than in a second subgroup of 7 body builders who had discontinued their intake of anabolic steroids at least 4 weeks prior to the study. In some single cases HDLC was barely detectable (2-7 mg.dl-1). The TG and TOTC remained unchanged. The present findings suggest that many years of body building among top-level athletes have no beneficial effect on lipoproteins and apolipoproteins. Simultaneous use of anabolic steroids results in part in extreme alterations in lipoproteins and apolipoproteins, representing an atherogenic profile. After discontinuing the use of anabolic steroids, the changes in lipid metabolism appear to be reversible.     

High density lipoprotein particle size distribution in subjects with obstructive jaundice

High density lipoproteins (HDL) from 14 patients with obstructive jaundice were examined by gradient gel electrophoresis to determine the effect of obstruction on particle size distribution. HDL from 7 of these patients were fractionated by gel permeation chromatography and further characterized by electron microscopy, SDS gel electrophoresis, apolipoprotein A-I and apolipoprotein A-II immunoturbidimetry, and analysis of chemical composition. In addition, lecithin:cholesterol acyltransferase (LCAT) activity was measured and correlated with plasma apolipoprotein A-I concentration and particle size distribution. HDL were abnormal in all patients regardless of severity, cause, or duration of obstruction. The major HDL subfraction in normal subjects, HDL3a (radius 4.1-4.3 nm) was either absent or considerably diminished, and HDL2b (radius 5.3 nm) was also frequently absent. Very small particles comparable in size to normal HDL3c (radius 3.8 nm) were prominent. In patients with a bilirubin concentration greater than 250 mumol/l, normal HDL had totally disappeared and were replaced by large discoidal particles of radius 8.5 nm and small spherical particles of radius 3.6-3.7 nm. Both populations of particles were markedly depleted of cholesteryl ester and enriched in free cholesterol and phospholipid. The discoidal particles were rich in apolipoproteins E, A-I, A-II, and C, while the small spherical particles contained predominantly apolipoprotein A-I. LCAT activity was diminished in all subjects to 8-54% of normal, and was strongly positively correlated (r = 0.91 P less than 0.05) with plasma apolipoprotein A-I levels.     

Distribution of cholesterol and apolipoprotein A-I and A-II in human high density lipoprotein subfractions separated by CsCl equilibrium gradient centrifugation: evidence for HDL subpopulations with differing A-I/A-II molar ratios.

The purpose of this experiment was to characterize the high density lipoproteins (HDL) as a function of hydrated density. HDL was subfractionated on the basis of hydrated density by CsCl density gradient centrifugation of whole serum or the d 1.063-1.25 g/ml HDL fraction isolated from three men and three women. Apolipoprotein A-I and A-II quantitation by radial immunodiffusion showed that the A-I/A-II ratio varied with the lipoprotein hydrated density. The A-I/A-II molar ratio of HDL lipoproteins banding between d 1.106 and 1.150 g/ml was nearly constant at 2.2 +/- 0.2. In the density range 1.151-1.25 g/ml the A-I/A-II ratio increased as the density increased. On the other hand, in the density range between 1.077 and 1.105 the A-I/A-II ratio increased as the density decreased, ranging from 2.8 +/- 0.5 for the d 1.093-1.105 g/ml fraction to 5.6 +/- 1.3 for the d 1.077-1.082 g/ml fraction. The d 1.063-1.076 g/ml fraction and the d 1.077-1.082 g/ml fractions had comparable A-I/A-II ratios. Serum and the d 1.063-1.25 g/ml HDL fraction exhibited similar trends. The cholesterol/(A-I + A-II) ratio decreased as the density increased in all 12 samples (six serum and six HDL) examined. Gradient gel electrophoresis of the density gradient fractions showed that as the density increased from 1.063 to 1.200 g/ml the apparent molecular weight decreased from 3.9 x 10(5) to 1.1 x 10(5). HDL subfractions with the same hydrated densities had comparable molecular weights and A-I/A-II and cholesterol/(A-I + A-II) ratios when isolated from men or women. HDL contains subpopulations that differ in the A-I/A-II molar ratio.-Cheung, M. C., and J. J. Albers. Distribution of cholesterol and apolipoprotein A-I and A-II in human high density lipoprotein subfractions separated by CsCl equilibrium gradient centrifugation: evidence for HDL subpopulations with differing A-I/A-II molar ratios.     

Genetic control of lipid transport in mice. II. Genes controlling structure of high density lipoproteins

Genetic factors controlling the structure of high density lipoproteins (HDL) in mice have been examined. Surveys of inbred strains of mice revealed genetic structural variations of the two major apolipoproteins of mouse HDL, apolipoproteins A-I and A-II. The structural variations alter the charge of the proteins as judged by isoelectric focusing of HDL under denaturing conditions. The structural variations are inherited as single Mendelian genes exhibiting co-dominant expression. The structural gene for mouse apolipoprotein A-II, designated Alp-2, resides on mouse chromosome 1, tightly linked to Ly-m20, a lymphocyte alloantigen locus. Previous studies, as well as our results, suggest that the structural gene for mouse apolipoprotein A-I, designated Alp-1, is on mouse chromosome 9. The genetic structural variation for apo-A-I results in a shift in the charge of the entire family of apo-A-I isoforms, indicating that they are all encoded by a common structural gene. The structure of intact HDL, examined primarily by electrophoretic techniques, exhibits numerous and complex phenotypes among different strains of mice. One variation, controlling the density and possibly the size of HDL, has been studied in two sets of recombinant inbred strains of mice. The results indicate that the variation is controlled by a single major gene that is either tightly linked to or identical with the Alp-2 gene on chromosome 1. In addition to structural variation, inbred strains of mice exhibited considerable quantitative variation of plasma HDL. Thus, the mouse provides a useful model system for examining the genetic control of mammalian HDL structure and regulation.     

Studies on the proteins involved in the interaction of high-density lipoprotein with isolated human small intestine epithelial cells.

Treatment of 125I-labelled high-density lipoprotein ([125I]HDL3) with monospecific polyclonal antibodies against apolipoproteins A-I and A-II resulted in a dose-dependent inhibition of the [125I]HDL3 binding to isolated human small intestine epithelial cells by 25% and 50%, respectively. Both antibodies also inhibited intracellular degradation of [125I]HDL3 by 80%. Treatment of enterocytes with polyclonal antibody against apolipoprotein A-I binding protein, a putative HDL receptor, inhibited both binding and degradation of [125I]HDL3 by these cells by 50%. Antibodies to apolipoprotein A-I, A-II and apo A-I-binding protein also inhibited [125I]HDL3 binding to cholesterol-loaded cells.     

Immunochemical characterization of two antigenic sites on human apolipoprotein A-I; localization and lipid modulation of these epitopes

Two monoclonal antibodies, A17 and A30, were raised against human apolipoprotein A-I (apo A-I). They were studied by competitive inhibition of 125I-labeled HDL3 with HDL subfractions, delipidated apo A-I, and complexes of dimyristoylphosphatidylcholine (DMPC) containing apo A-I and apo A-II. Immunoblotting located the A17 antibody on CNBr fragment 4 of apo A-I and the A30 antibody on CNBr fragment 1. The A17 antigenic determinant was expressed identically in all HDL subclasses, on delipidated apo A-I as well as all on the DMPC-apo A-I and DMPC-apo A-I/apo A-II complexes. In contrast, the apparent affinity constant of the A30 antibody for delipidated apo A-I was about 30-times less than for HDL3 or for apo A-I/apo A-II-phospholipid complexes. These data suggest that the association of apo A-I with phospholipids improves the reactivity of the A30 monoclonal antibody towards apo A-I, and that this antigenic determinant has a different conformation in delipidated apo A-I compared to apo A-I complexed with phospholipids. Turbidimetric and fluorescence experiments monitoring the phospholipid-apo A-I association in the presence and in the absence of the A17 and A30 antibodies were consistent with the competition experiments carried out by solid phase radioimmunoassay (RIA). After reaction of apo A-I with the A30 antibody, we observed an enhancement of the degradation kinetics of large multilamellar vesicles (LMV), while the A17 antibody did not have a significant effect. Calcein leakage experiments carried out below the transition temperature of DPPC showed an enhancement of the degradation kinetics with both monoclonal antibodies, while the phase-transition release was independent of the reaction of apo A-I with the monoclonal antibodies. These data therefore suggest the existence of at least two different types of epitope on apo A-I, which might account for the differences in immunological reactivity of apo A-I that is either delipidated or present on HDL.     

A purification method for apolipoprotein A-I and A-II

Apolipoproteins A-I and A-II were isolated from precipitates obtained by cold ethanol fractionation of human plasma. The starting material used in this report was precipitate B of the Kistler and Nitschmann method which corresponds approximately to fraction III of the Cohn and Oncley procedure. Through the use of urea, chloroform, and ethanol in appropriate concentrations, apolipoproteins A-I and A-II were isolated by a simple extraction technique avoiding time-consuming ultracentrifugation. Starting from 10 g of centrifuged precipitate B, approximately 100 mg of apolipoprotein A-I and 10 mg of apolipoprotein A-II were obtained. When incubated with normal human or rabbit plasma, both apolipoproteins were readily incorporated into high-density lipoproteins. Apolipoprotein A-I obtained by the cold ethanol method activated lecithin-cholesterol acyltransferase to the same extent as apolipoprotein A-I prepared by the classical flotation method. Apolipoprotein A-II had no such properties by itself, but was capable of potentiating lecithin-cholesterol acyltransferase activity of apolipoprotein A-I.     

Stable isotope turnover of apolipoproteins of high-density lipoproteins in humans

Amino acid precursors labelled with stable isotopes have been successfully used to explore the metabolism of the apolipoproteins of HDL. Some methodological and mathematical modelling problems remain, mainly related to amino acid recycling in a plasma protein such as apolipoprotein A-I with a long residence time (the reciprocal of the fractional catabolic rate) of 4-5 days. Apolipoprotein A-I, apolipoprotein E, and apolipoprotein A-IV in triglyceride-rich lipoproteins (containing chylomicrons, VLDL, and remnants) exhibit more complex kinetics. The small amounts of apolipoprotein A-I and of apolipoprotein A-IV in the triglyceride-rich lipoproteins have a residence time similar to that of the apolipoprotein A-I of HDL. In contrast, the apolipoprotein E in triglyceride-rich lipoproteins has been found to have an average residence time of 0.11 days. Diets low in saturated fat and cholesterol, which lower HDL levels, do so by decreasing the secretion of apolipoprotein A-I, with apolipoprotein A-II kinetics unaffected. Individuals with impaired glucose tolerance have a decreased residence time of apolipoprotein A-I but no change in secretion rate or in apolipoprotein A-II kinetics. This suggests a link between insulin resistance and the risk of atherosclerosis. In heterozygous familial hypercholesterolemia, both the fractional catabolic rate and the secretion rate of apolipoprotein A-I are increased, resulting in no change in the plasma level. Stable isotope studies have strengthened the evidence that triglyceride enrichment of HDL increases its catabolism Laboratory.     

On the structural similarity of ApoA-I and ApoA-II of human high density lipoproteins.

The possible evolutionary origin of apolipoproteins was studied by comparing the primary structures of different plasma apolipoproteins and other phospholipid-binding proteins. Apolipoprotein A-I (ApoA-I) and apolipoprotein A-II (ApoA-II) of human high density lipoprotein (HDL) are related. The resemblance of these two HDL apolipoproteins are apparently restricted to the carboxyl terminal regions suggesting that these portions of the molecules are derived from the same ancestor. The homologous carboxyl terminal segments may be involved in the regulation of HDL metabolism or in the interaction with phospholipids.     

Difference in lipid packing sensitivity of exchangeable apolipoproteins apoA-I and apoA-II: an important determinant for their distinctive role in lipid metabolism

Exchangeable apolipoproteins A-I and A-II play distinct roles in reverse cholesterol transport. ApoA-I interacts with phospholipids and cholesterol of the cell membrane to make high density lipoprotein particles whereas apolipoprotein A-II interacts with high density lipoprotein particles to release apolipoprotein A-I. The two proteins show a high activity at the aqueous solution/lipid interface and are characterized by a high content of amphipathic α-helices built upon repetition of the same structural motif. We set out to investigate to what extent the number of α-helix repeats of this structural motif modulates the affinity of the protein for lipids and the sensitivity to lipid packing. To this aim we have compared the insertion of apolipoproteins A-I and A-II in phospholipid monolayers formed on a Langmuir trough in conditions where lipid packing, surface pressure and charge were controlled. We also used atomic force microscopy to obtain high resolution topographic images of the surface at a resolution of several nanometers and performed statistical image analysis to calculate the spatial distribution and geometrical shape of apolipoproteins A-I and A-II clusters. Our data indicate that apolipoprotein A-I is sensitive to packing of zwitterionic lipids but insensitive to the packing of negatively charged lipids. Interestingly, apolipoprotein A-II proved to be insensitive to the packing of zwitterionic lipids. The different sensitivity to lipid packing provides clues as to why apolipoprotein A-II barely forms nascent high density lipoprotein particles while apolipoprotein A-I promotes their formation. We conclude that the different interfacial behaviors of apolipoprotein A-I and apolipoprotein A-II in lipidic monolayers are important determinants of their distinctive roles in lipid metabolism.     

Effect of high-density lipoproteins with varying ratios of apolipoprotein A-I to apolipoprotein A-II on steroidogenesis by cultured rat ovary granulosa cells

Plasma high-density lipoproteins (HDL) can provide rat ovary steroidogenic tissue with cholesterol for steroid hormone production, but the mechanism of cholesterol transfer is unknown. To test the importance of apolipoprotein A-I (the major HDL apolipoprotein) in HDL-cell interactions, we examined the ability of canine-human HDL hybrids containing various proportions of canine apolipoprotein A-I and human apolipoprotein A-II to stimulate steroidogenesis by cultured rat ovary granulosa cells. We observed that as the apolipoprotein A-II to apolipoprotein A-II ratio decreased, the ability of the hybrid particles to stimulate granulosa cell progestin (progesterone and 20 alpha-dihydroprogesterone) production diminished. However, granulosa cell progestin (progesterone and 20 alpha-dihydroprogesterone) production diminished. However, apolipoprotein A-I was not necessary for cholesterol transfer, since hybrids with less than 5% of their total apolipoprotein mass as apolipoprotein A-I stimulated progestin production 30% as effectively as canine HDL, which contained essentially only apolipoprotein A-I. These data indicate that the delivery of cholesterol from HDL into the rat ovary cell for steroidogenesis is not strictly dependent on the presence of a specific HDL apolipoprotein.