LDL and cAMP cooperate to regulate the functional expression of the LRP in rat ovarian granulosa cells

Rat ovarian granulosa rely heavily on lipoprotein-derived cholesterol for steroidogenesis, which is principally supplied by the LDL receptor- and scavenger receptor class B type I (SR-BI)-mediated pathways. In this study, we characterized the hormonal and cholesterol regulation of another member of the LDL receptor superfamily, low density lipoprotein receptor-related protein (LRP), and its role in granulosa cell steroidogenesis. Coincubation of cultured granulosa cells with LDL and N6,O2'-dibutyryl adenosine 3',5'-cyclic monophosphate (Bt2cAMP) greatly increased the mRNA/protein levels of LRP. Bt2cAMP and Bt2cAMP plus human hLDL also enhanced SR-BI mRNA levels. However, there was no change in the expression of receptor-associated protein, a chaperone for LRP, or another lipoprotein receptor, LRP8/apoER2, in response to Bt2cAMP plus hLDL, whereas the mRNA expression of LDL receptor was reduced significantly. The induced LRP was fully functional, mediating increased uptake of its ligand, alpha2-macroglobulin. The level of binding of another LRP ligand, chylomicron remnants, did not increase, although the extent of remnant degradation that could be attributed to the LRP doubled in cells with increased levels of LRP. The addition of lipoprotein-type LRP ligands such as chylomicron remnants and VLDL to the incubation medium significantly increased the progestin production under both basal and stimulated conditions. In summary, our studies demonstrate a role for LRP in lipoprotein-supported ovarian granulosa cell steroidogenesis.     

Human hepatic low-density lipoprotein receptors: associations of receptor activities in vitro with plasma lipid and apolipoprotein concentrations in vivo

The relationships of plasma lipid and apolipoprotein (apo) concentrations to hepatic low-density lipoprotein (LDL) receptor activity were examined in 21 subjects (16 females, 5 males), who were undergoing laparotomy for non-neoplastic disease (cholecystectomy in 16). None had familial hypercholesterolemia, or renal, endocrine or hepatic disease. Ages were 37-77 years (mean, 58 years), plasma cholesterol concentrations 4.09-6.72 mmol/l (5.38) and plasma triacylglycerol concentrations 0.75-2.35 mmol/l (1.36). Receptor activity was quantified in vitro as the total saturable binding and EDTA-suppressible binding (representing apoB,E receptors) of 125I-labelled human LDL (15 micrograms protein/ml) by liver homogenate at 37 degrees C. There were no significant differences between men and women in 125I-labeled LDL binding. In the pooled data, EDTA-suppressible binding averaged 50 ng 125I-LDL protein/mg cell protein (S.D., 15). Total saturable binding averaged 2-fold greater (mean, 101 ng/mg; S.D., 32). Plasma cholesterol, LDL cholesterol and apoB concentrations were negative functions of both EDTA-suppressible binding and total saturable binding, but the correlations with EDTA-suppressible binding were stronger (cholesterol: r = -0.59, P less than 0.01; LDL cholesterol: r = -0.48, P less than 0.05; apoB: r = -0.61, P less than 0.01). Plasma triacylglycerol, high-density lipoprotein cholesterol and apoA-I concentrations were not related to either measure of receptor activity. These results provide evidence that the activity of apoB,E receptors in the liver is a major determinant of the plasma LDL concentration in middle-aged and elderly humans.     

Effect of cell cholesterol content on apolipoprotein B secretion and LDL receptor activity in the human hepatoma cell line, HepG2

Human hepatoma HepG2 cells were used to study the effects of cholesterol loading and depletion on apolipoprotein B (apoB) secretion and low-density lipoprotein (LDL) receptor activity. Exposure of HepG2 cells to cholesterol and oleic acid, which elevated intracellular cholesterol levels, stimulated apoB secretion and reduced receptor-mediated uptake of LDL, whereas recombinant complexes of apolipoprotein A-I with dimyristoylphosphatidylcholine, which depleted the cellular cholesterol pool, inhibited apoB secretion and up-regulated LDL receptors. Significant negative correlation (r = -0.92, P less than 0.001) between the levels of apoB secretion and LDL uptake was found. These data suggest that the cholesterol content of the cells may induce concomitant changes in apoB secretion and LDL receptor activity.     

Apolipoprotein B: its role in the control of fibroblast cholesterol biosynthesis and in the regulation of its own binding to cellular receptors.

Apolipoprotein B transports cholesterol in plasma as low density lipoprotein (LDL) and targets its delivery to cells by binding to a specific plasma membrane receptor. The cellular consequences of apoB binding to its receptor were investigated to determine whether it suppresses cholesterol biosynthesis and reduces the number of cellular receptors for the apoprotein. Upon preincubation of fibroblasts with lipoprotein-deficient medium alone or supplemented with either LDL or apoB complexed to BSA (apoB-BSA), LDL suppressed cholesterol biosynthesis, but apoB enhanced it. Similarly, fibroblasts preincubated in medium supplemented with LDL bound decreased amounts of either (125)I-labeled LDL or (125)I-labeled apoB-BSA to their receptors, while preincubation with apoB-BSA increased the binding relative to the controls. These latter results occurred in association with a decrease in cellular cholesterol content, indicating that apoB in the medium bound cholesterol and removed it from the cells, thus stimulating both cholesterol synthesis and cellular binding of apoB. Accordingly, fibroblast cholesterol synthesis and the number of functional LDL receptors are not suppressed by the binding of the apoprotein to the receptor, and the known role of apoB remains that of transporting cholesterol in plasma and delivering it to the cell. A possible physiologic role for apoB in depleting cells of cholesterol is presently unknown since apoB is not known to exist free in plasma; however, these findings demonstrate such a functional capability for this apoprotein.-Shireman, R. B., and W. R. Fisher. Apolipoprotein B: its role in the control of fibroblast cholesterol biosynthesis and in the regulation of its own binding to cellular receptors.     

Effects of dietary cholesterol and fatty acids on plasma cholesterol level and hepatic lipoprotein metabolism

The effects of dietary cholesterol and fatty acids on the plasma cholesterol level and rates of very low density lipoprotein (VLDL) cholesterol secretion and low density lipoprotein (LDL) transport through LDL receptors in the liver of the hamster were investigated. Increases of plasma VLDL- and LDL-cholesterol levels and VLDL-cholesterol secretion from hepatocytes were observed in animals fed a diet enriched with 0.1% cholesterol for 2 weeks in comparison with animals fed a control diet. The addition of dietary palmitic acid accelerated the effect of dietary cholesterol on plasma VLDL- and LDL-cholesterol levels and VLDL-cholesterol secretion from hepatocytes. Dietary linoleic acid accelerated the effect of dietary cholesterol on VLDL-cholesterol secretion from hepatocytes and diminished the effect on the plasma LDL-cholesterol level. Hepatic LDL receptor activity was considerably suppressed by a control diet containing 0.05% cholesterol and a further small suppression was induced by a diet enriched with 0.1% cholesterol with or without 5% palmitic acid. However, dietary linoleic acid diminished the effect of dietary cholesterol on the suppression of hepatic LDL receptor activity. These results suggest that dietary palmitic acid augments the effect of dietary cholesterol in elevating the plasma LDL-cholesterol level through acceleration of VLDL-cholesterol secretion from the liver, and that dietary linoleic acid diminishes the effect of dietary cholesterol in elevating the plasma LDL-cholesterol level by preventing the suppression of hepatic LDL receptor activity induced by cholesterol.     

A novel and simple method for quantification of small,dense LDL

A preponderance of small, dense (sd) LDL is strongly associated with the development of coronary heart disease, but the method for the measurement of sd LDL is too laborious for clinical use. We report a simple method for the quantification of sd LDL that is applicable to an autoanalyzer. This method consists of two steps: first, to precipitate the lipoprotein of density (d) <1.044 g/ml using heparin-magnesium; and second, to measure LDL-cholesterol in the supernatant by the homogeneous method or apolipoprotein B (apoB) by an immunoturbidometric assay. The cholesterol and apoB values obtained by the precipitation method (45 +/- 26 and 33 +/- 20 mg/dl, respectively) were similar to those obtained in the lipoprotein (d = 1.044-1.063) separated by ultracentrifugation (42 +/- 22 and 31 +/- 17 mg/dl, respectively), and there was an excellent correlation between the two methods for sd LDL-cholesterol (y = 1.05X + 1, r = 0.88, n = 69) and apoB (y = 1.07X, r = 0.90). Sd LDL values had a significant inverse correlation with LDL size. A high correlation was found between sd LDL-cholesterol and apoB values (r = 0.94). Sd LDL value was related to triglyceride, apoB, and LDL-cholesterol, but not to the buoyant LDL level. These results suggest that this precipitation method is a simple and rapid method for the measurement of sd LDL concentration.     

Use of low-density and high-density lipoproteins in undifferentiated porcine granulosa cells

Granulosa cells aspirated from medium-sized porcine ovarian follicles (3-5 mm) in short-term incubation responded to the addition of both low-density lipoprotein (LDL) and high-density lipoprotein (HDL) with increased accumulation of progesterone. HDL was more potent than LDL in enhancing progesterone secretion. When granulosa cells were cultured under serum-free conditions for 72 h, HDL but not LDL exhibited a dose-dependent enhancement of progesterone secretion. Addition of insulin to the cells greatly potentiated the stimulatory effect of LDL on progesterone accumulation, while the response to HDL was only slightly increased. Granulosa cells in culture degraded LDL but not HDL. Addition of insulin enhanced LDL degradation. Exposure of cells in culture to chloroquine, an inhibitor of lysosomal function, completely blocked LDL degradation and LDL-induced stimulation of steroidogenesis. The stimulatory effect of HDL was not affected by chloroquine. We interpret these findings to indicate that granulosa cells derive cholesterol from LDL by means of lysosomal degradation, which is not required for use of cholesterol from HDL. Monensin, a carboxylic ionophore that interrupts recycling of LDL receptors, prevented LDL-enhanced progesterone accumulation but not HDL-induced stimulation. This provides evidence that HDL-induced stimulation of steroidogenesis does not involve LDL receptors. We conclude that HDL present in follicular fluid is capable of providing cholesterol to granulosa cells for steroidogenesis. The stimulatory effect of HDL is not due to the presence of apoprotein E, an HDL component that binds to the LDL receptor. A unique HDL pathway that does not involve LDL receptors and lysosomal degradation may operate in porcine granulosa cells.     

Characterization of lipoprotein receptors on rat Fu5AH hepatoma cells

The rat hepatoma cell line Fu5AH has the unusual property of accumulating massive amounts of cholesteryl ester upon incubation with hypercholesterolemic serum, and especially when incubated with beta-very low density lipoproteins (beta-VLDL) from cholesterol-fed dogs. The present study was designed to identify and characterize the lipoprotein receptors that mediate the cholesteryl ester accumulation. The beta-VLDL and cholesterol-induced apolipoprotein (apo) E-containing high density lipoproteins (apoE HDLc) bound to Fu5AH cells with very high affinity (Kd approximately equal to 10(-10) M), whereas low density lipoproteins (LDL) bound with unusually low affinity (Kd approximately equal to 10(-8) M). Receptor binding activity of 125I-labeled beta-VLDL, 125I-labeled apoE HDLc, and 125I-labeled LDL was abolished by incubation in the presence of an excess of unlabeled LDL or of a polyclonal antibody to the bovine adrenal apoB,E(LDL) receptor. The receptors were completely down-regulated by preincubating Fu5AH cells with beta-VLDL, but much higher levels of beta-VLDL were required than for down-regulation of fibroblast apoB,E(LDL) receptors. Receptor binding was abolished by reductive methylation of the lysyl residues of the apolipoprotein of the beta-VLDL and by an apoE monoclonal antibody (1D7) that blocks receptor binding. The Fu5AH receptor was further characterized by using the bovine adrenal apoB,E(LDL) receptor antibody. A single protein (Mr approximately equal to 130,000) was identified in Triton extracts of whole cells, and two proteins (Mr approximately equal to 130,000 and 115,000) were found in Fu5AH cell membranes disrupted by homogenization. The Mr approximately equal to 115,000 protein was released from the membranes and did not react with an antibody to the carboxyl-terminal (cytoplasmic) domain of the apoB,E(LDL) receptors. These studies indicate that Fu5AH cells express apoB,E(LDL) receptors that have unusually low affinity for apoB-continuing lipoproteins, require large amounts of cholesterol to induce down-regulation, and are susceptible to specific proteolysis in cell homogenates. These apoB,E(LDL) receptors are responsible for the receptor-mediated uptake of beta-VLDL and chylomicron remnants by Fu5AH cells.     

The beta very low density lipoprotein present in hepatic lipase deficiency competitively inhibits low density lipoprotein binding to fibroblasts and stimulates fibroblast acyl-CoA:cholesterol acyltransferase

Beta very low density lipoprotein (VLDL) was isolated from a patient with hepatic lipase deficiency. The particles were found to contain apolipoprotein B-100 (apoB) and apolipoprotein E (apoE) and were rich in cholesterol and cholesteryl ester relative to VLDL with pre beta electrophoretic mobility. These particles were active in displacing human low density lipoprotein (LDL) from the fibroblast apoB,E receptor and produced a marked stimulation of acyl-CoA:cholesterol acyltransferase. Treatment of intact beta-VLDL with trypsin abolished its ability to displace LDL from fibroblasts. Incubation of trypsin treated beta-VLDL with fibroblasts resulted in a significant stimulation of acyl-CoA:cholesterol acyltransferase activity. beta-VLDL isolated from a patient with Type III hyperlipoproteinemia and an apoE2/E2 phenotype had a higher cholesteryl ester/triglyceride ratio than the beta-VLDL of hepatic lipase deficiency and contained apoB48. It displaced LDL from fibroblasts to a small but significant extent. The Type III beta-VLDL stimulated acyl-CoA:cholesterol acyltransferase to a level similar to that of trypsin-treated beta-VLDL isolated from the hepatic lipase-deficient patient. These results demonstrate that the cholesterol-rich beta-VLDL particles present in patients with hepatic lipase deficiency are capable of interacting with fibroblasts via the apoB,E receptor and that this interaction is completely due to trypsin-sensitive components of the beta-VLDL. These particles were very effective in stimulating fibroblast acyl-CoA:cholesterol acyltransferase. This stimulation was due to both trypsin-sensitive and trypsin-insensitive components.     

Studies on the expression of genes encoding apolipoproteins B100 and B48 and the low density lipoprotein receptor in nonhuman primates. Comparison of dietary fat and cholesterol

African green monkeys were fed diets containing low and moderate cholesterol concentrations with either polyunsaturated or unsaturated fat as 40% of calories. Plasma total cholesterol, low density lipoprotein (LDL) cholesterol, and apoB concentrations generally were higher in animals fed (a) the higher dietary cholesterol concentration and (b) saturated fat. At necropsy, liver and intestine were removed, and measurement of mRNAs for LDL receptors (liver) and for apolipoprotein B (liver and intestine) was done. Monkey small intestine mucosa made exclusively apoB48 while the liver made only apoB100, although apoB mRNA in both tissues was the same size (14 kilobases). No dietary cholesterol or fat effects were found for apoB mRNA abundance in the liver, while the animals fed the higher dietary cholesterol level had 50% lower levels of hepatic LDL receptor mRNA. In a separate group of animals, livers were perfused and the rate of apoB secretion was measured. No dietary fat effect on apoB secretion rate was found, and no relationship between plasma LDL cholesterol concentration and the rate of hepatic apoB production existed. These findings support the idea that the dietary factors that increase LDL concentrations act by reducing clearance of apoB-containing particles rather than by increasing production of these lipoproteins. Hepatic LDL receptor mRNA was similar in abundance in polyunsaturated fat and saturated fat-fed animals, suggesting that the difference in plasma cholesterol concentration between these groups is not mediated via effects on LDL receptor mRNA abundance. The level of intestinal apoB mRNA was about 30% higher in animals fed the moderate dietary cholesterol concentration. Earlier studies have shown that more cholesterol is transported in chylomicrons from the intestine when dietary cholesterol levels are higher, and the increased intestinal apoB mRNA abundance may reflect increased intestinal cholesterol transport and chylomicron apoB48 production.     

The composition, structural properties and binding of very-low-density and low-density lipoproteins to the LDL receptor in normo- and hypertriglyceridemia: relation to the apolipoprotein E phenotype

The composition, apolipoprotein structure and lipoprotein binding to the LDL receptor were studied for very-low-density (VLDL) and low-density lipoprotein (LDL) particles isolated from subjects with apoE phenotype E3/3 (E3), E2/2 or E2/3 (E2+) and E3/4 or E4/4 (E4+) and a wide range of plasma triglyceride (TG) contents. The data combined for all three phenotype groups can be summarized as follows. (i) A decrease in accessibility of VLDL tryptophan residues to I- anions with a decrease in tryptophan surface density, concomitant with an increase in VLDL dimensions, reflects the increased efficiency of protein-protein interactions. (ii) A gradual increase in the quenching constant for LDL apoB fluorescence with an increase in TG/cholesterol (Chol) ratio reflects the 'freezing' effect of Chol molecules on apoB dynamics. (iii) Different mechanisms specific for a particular lipoprotein from E3/3 or E2/3 subjects are responsible for apoE-mediated VLDL binding and apoB-mediated LDL binding to the LDL receptor in a solid-phase binding assay. (iv) The 'spacing' effect of apoC-III molecules on apoE-mediated VLDL binding results in a decrease in the number of binding sites. (v) The maximum of the dependence of the LDL binding affinity constant on relative tryptophan density corresponds to LDL intermediate size. VLDL particles from hypertriglyceridemic E2/3 heterozygotic individuals had remnant-like properties (increased cholesterol, apoE and decreased apoC-III content) while their binding efficiency was unchanged. Based on the affinity constant value and LDL-Chol content, increased competition between VLDL and LDL for the binding to the LDL receptor upon increase in plasma TG is suggested, and LDL from hypertriglyceridemic E3/3 homozygotic individuals is the most efficient competitor.     

Regulation of steroidogenesis and cholesterol synthesis by prostaglandin F-2 alpha and lipoproteins in bovine luteal cells

Bovine luteal cells can utilize low density lipoprotein (LDL) or high density lipoprotein (HDL) as a source of cholesterol for steroidogenesis, and administration of PGF-2 alpha in vitro suppresses lipoprotein utilization. The objective of this study was to examine the mechanism by which PGF-2 alpha exerts this effect. Cultured bovine luteal cells received 0.25 microCi[14C]acetate/ml, to assess rates of de-novo sterol and steroid synthesis, with or without lipoproteins. Both LDL and HDL enhanced progesterone production (P less than 0.01), but caused a significant reduction in the amount of radioactivity in the cholesterol fraction. PGF-2 alpha treatment inhibited the increase in lipoprotein-induced progesterone synthesis (P less than 0.01), but did not prevent the reduction in de-novo cholesterol synthesis brought about by LDL or HDL. PGF-2 alpha alone reduced cholesterol synthesis (P less than 0.01), but it was not as effective as either LDL or HDL. Both lipoproteins and PGF-2 alpha also decreased the amount of radioactivity in the progesterone fraction (P less than 0.01), and the effect of PGF-2 alpha was similar to that of the lipoproteins. It is concluded that lipoproteins can enhance progesterone production and also suppress de-novo cholesterol synthesis in bovine luteal cells, but only the former effect of lipoproteins is inhibited by PGF-2 alpha. Therefore, it is suggested that PGF-2 alpha allows entry of lipoprotein cholesterol into the cell, but prevents utilization for steroidogenesis. In addition, PGF-2 alpha alone can suppress cholesterol synthesis, as well as decrease conversion of cholesterol to progesterone.     

Apolipoprotein E Structure and Substrate and Receptor-Binding Activities of Triglyceride-Rich Human Plasma Lipoproteins in Normo- and Hypertriglyceridemia

Cysteine-arginine interchanges along the primary sequence of human plasma apolipoprotein E (apoE) play an important role in determining its biological functions due to a high mutation frequency of cytosine in CGX triplet that codes 33 of 34 apolipoprotein arginine residues. The contribution of apoE secondary structure to apolipoprotein-lipid interaction is described. The significance of apolipoprotein in triglyceride synthesis, lipoprotein lipolysis, and receptor-mediated clearance of lipolytic remnants of triglyceride-rich lipoproteins is discussed as well. The metabolic flow of lipoproteins in normo- and hypertriglyceridemia can be described by separate compartments that contribute to lipoprotein interaction with at least six different receptors: 1) low density lipoprotein (LDL) receptor; 2) LDL receptor-related protein (LRP); 3) apoB(48) macrophage receptor for hypertriglyceridemic very low density lipoproteins (VLDL); 4) scavenger receptors; 5) VLDL receptor; 6) lipolysis-stimulated receptor. The contribution of the exposure of apoE molecules on the surface of triglyceride-rich particles sensitive both to lipolysis and plasma triglyceride content to the interaction with LDL receptor and LRP is emphasized.     

The influence of the triglyceride content of low density lipoprotein on the interaction of apolipoprotein B-100 with cells

To study the effect of triglyceride content of low density lipoprotein (LDL) on its physicochemical and biological properties, we have depleted the triglyceride by incubation with hepatic lipase (HL-LDL) and raised the triglyceride by incubation of HL-LDL with very low density lipoprotein and lipoprotein-deficient serum. HL-LDL was taken up by human monocyte-derived macrophages and by human skin fibroblasts at an increased rate compared to untreated LDL. Incubation of the various LDL preparations revealed that cellular LDL degradation as well as LDL-mediated cholesterol esterification were inversely related to the triglyceride content of the LDL preparation. Modification of the triglyceride content of LDL also was associated with changes in the free fatty acid content, but the interaction of the LDL with cells was unaffected by the level of this component. The triglyceride content of LDL was found to be reciprocally related to the number of free lysine amino groups of LDL apolipoprotein B (apoB) which could be labeled with trinitrobenzenesulfonic acid. 13C-Nuclear magnetic resonance (NMR) spectra of native LDL and HL-LDL samples containing [13CH3]2 lysine residues formed by reductive methylation (11-13% modification) showed that the arrangement of apoB lysines is perturbed by the exposure to hepatic lipase. The ratio of labeled lysines with pK 8.9 to those with pK 10.5 exposed on the surface of LDL particles was decreased by about 40% by lipase treatment. These effects are apparently due to changes in local apoB conformation because circular dichroism spectra revealed that the average secondary structure of the entire apoB molecule is the same in native LDL and HL-LDL. The triglyceride content of LDL reciprocally affected its binding to a monoclonal antibody which recognizes epitopes around the LDL receptor binding domain of apoB. The above evidence indicates that modulation of the core triglyceride and possibly also surface phospholipid content of LDL can alter the conformation of apoB on the surface of the particle, thereby influencing the interaction with cell surface LDL receptors.     

ELISA quantitation of apolipoproteins in plasma lipoprotein fractions: ApoE in apoB-containing lipoproteins (Lp B:E) and apoB in apoE-containing lipoproteins (Lp E:B)

Growing clinical evidence suggests that metabolic behavior and atherogenic potential vary within lipoprotein subclasses that can be defined by apolipoprotein variation. Variant constituency of apolipoproteins B and E (apoB and apoE) may be particularly important because of the central roles of these apolipoproteins in the endogeneous lipid delivery cascade. ApoB is the sole protein of low-density lipoprotein (LDL), and like LDL cholesterol, the plasma apoB level has been positively correlated with risk for atherosclerotic disease. ApoE is a major functional lipoprotein in the triglyceride-rich lipoproteins, and may be crucial in the conversion of very low density lipoprotein (VLDL) to LDL. Based on work by others that enabled the quantititation of apoB-containing particles by content of up to two other types of apolipoprotein, we have developed a method for determining the amount of apoE in apoB-containing lipoproteins (Lp B:E) and the amount of apoB in apoE-containing lipoproteins (Lp E:B). From the Lp B:E and Lp E:B concentrations, the molar ratio of apoE to apoB in lipoproteins containing apoB and/or apoE in plasma can be determined. The methodology is fast, specific, and sensitive and should prove extremely useful in further categorizing lipoproteins and characterizing their behavior. In applying this method to clinical groupings of normo- and hyperlipidemia, we found that the plasma triglyceride level correlated with the apoE and Lp B:E concentrations in plasma, while the total cholesterol level correlated with the apoB and Lp E:B levels.     

Lipoprotein receptor expression during luteinization of the ovarian follicle

Ovarian follicles luteinize after ovulation, requiring structural and molecular remodeling along with exponential increases in steroidogenesis. Cholesterol substrates for luteal steroidogenesis are imported via scavenger receptor-BI (SR-BI) and the low-density lipoprotein (LDL) receptor from circulating high-density lipoproteins and LDL. SR-BI mRNA is expressed in pig ovaries at all stages of folliculogenesis and in the corpus luteum (CL). An 82-kDa form of SR-BI predominates throughout, is weakly present in granulosa cells, and is robustly expressed in the CL, along with the less abundant 57-kDa form. Digestion of N-linked carbohydrates substantially reduced the SR-BI mass in luteal cells, indicating that differences between forms is attributable to glycosylation. Immunohistochemistry revealed SR-BI to be concentrated in the cytoplasm of follicular granulosa cells, although found mostly at the periphery of luteal cells. To examine receptor dynamics during gonadotropin-induced luteinization, pigs were treated with an ovulatory stimulus, and ovaries were collected at intervals to ovulation. SR-BI in granulosa cell cytoplasm increased through the periovulatory period, with migration to the cell periphery as the CL matured. In vitro culture of follicles with human chorionic gonadotropin induced time-dependent upregulation of 82-kDa SR-BI in granulosa cells. SR-BI and LDL receptor were reciprocally expressed, with the latter highest in follicular granulosa cells, declining precipitously with CL formation. We conclude that luteinization causes upregulation of SR-BI expression, its posttranslational maturation by glycosylation, and insertion into luteal cell membranes. Expression of the LDL receptor is extinguished during luteinization, indicating dynamic regulation of cholesterol importation to maintain elevated steroid output by the CL.     

Uptake of gold- and [3H]cholesteryl linoleate-labeled human low density lipoprotein by cultured rat granulosa cells: cellular mechanisms involved in lipoprotein metabolism and their importance to steroidogenesis

We used electron microscopy, acid hydrolase cytochemistry, and biochemistry to analyze the uptake and metabolism of colloidal gold- and [3H]cholesteryl linoleate-labeled human low density lipoprotein (LDL) by cultured rat granulosa cells. The initial interaction of gold-LDL conjugates with granulosa cells occurred at binding sites diffusely distributed over the plasma membrane. After incubation with ligand in the cold, 99.9% of the conjugates were at the cell surface but less than 4% lay over coated pits. Uptake was specific since it was decreased 93-95% by excess unconjugated LDL and heparin, but only 34-38% by excess unconjugated human high density lipoprotein. LDL uptake was related to granulosa cell differentiation; well-luteinized cells bound 2-3 times as much gold-LDL as did poorly luteinized cells. Ligand internalization was initiated by warming and involved coated pits, coated vesicles, pale multivesicular bodies (MVBs), dense MVBs, and lysosomes. A key event in this process was the translocation of gold-LDL conjugates from the cell periphery to the Golgi zone. This step was carried out by the pale MVB, a prelysosomal compartment that behaves like an endosome. Granulosa cells exposed to LDL labeled with gold and [3H]cholesteryl linoleate converted [3H]sterol to [3H]progestin in a time-dependent manner. This conversion was paralleled by increased gold-labeling of lysosomes and blocked by chloroquine, an inhibitor of lysosomal activity. In brief, granulosa cells deliver LDL to lysosomes by a receptor-mediated mechanism for the hydrolysis of cholesteryl esters. The resulting cholesterol is, in turn, transferred to other cellular compartments, where conversion to steroid occurs. These events comprise the pathway used by steroid-secreting cells to obtain the LDL-cholesterol vital for steroidogenesis.     

Receptor binding activity of lipid recombinants of apolipoprotein B-100 thrombolytic fragments

Apolipoprotein (apo) B-100, the protein constituent of low density lipoproteins (LDL), is the determinant responsible for LDL binding to the apoB,E(LDL) receptor on cells. The current study was designed to identify the region(s) of apoB-100 that interact with the apoB,E(LDL) receptor. Apolipoprotein B-100 was fragmented by thrombin digestion, and the isolated fragments (T2, T3, T4) were recombined with cholesterol-induced canine high density lipoproteins (HDLc). Before the recombination, the receptor binding activity of apoE of the HDLc was abolished by reductive methylation and extensive trypsin treatment. This treatment permitted almost complete replacement of the small residual apoE fragments by the large apoB fragments. Recombinant apoB particles were isolated by ultracentrifugation and tested for binding to receptors on cultured human fibroblasts. The recombinant particles had chemical and physical properties similar to those of native HDLc. Recombinants of both the whole thrombolytic digest and of isolated fragments displayed specific binding to the apoB,E (LDL) receptor. Anti-apoB,E(LDL) receptor antibodies abolished 90% of the binding, and there was almost no specific binding to receptor-negative fibroblasts or to cells in which the receptors had been down-regulated. The binding of apoB-100 recombinants to the receptor also demonstrated calcium dependency; in addition, the surface binding of the recombinants was released by polyanionic compounds. All these recombinants had binding affinities comparable to one another but less than that of native LDL. Although T2, T3 and T4 recombinants can all bind specifically to the apoB,E(LDL) receptor, it remains to be established whether their activity represents physiologically relevant binding. Nevertheless, the present findings illustrate the potential of the recombinant method using HDLc lipids to reconstitute biological activity.