Low density lipoprotein receptors in bovine adrenal cortex. I. Receptor-mediated uptake of low density lipoprotein and utilization of its cholesterol for steroid synthesis in cultured adrenocortical cells.

0013-7227/79/1043-0599S02.00 Endocrinology Copyright © 1979 by The Endocrine Society

Vol. 104, No. 3 Printed in U.S.A.

Low Density Lipoprotein Receptors in Bovine Adrenal Cortex. I. Receptor-Mediated Uptake of Low Density Lipoprotein and Utilization of Its Cholesterol for Steroid Synthesis in Cultured Adrenocortical Cells* PETRI T. KOVANEN.f JERRY R. FAUST, MICHAEL S. BROWN, AND JOSEPH L. GOLDSTEIN Departments of Molecular Genetics and Internal Medicine, University of Texas Health Science Center at Dallas, Dallas, Texas 75235

ABSTRACT. Functioning bovine adrenocortical cells in monolayer culture were shown to obtain cholesterol for steroid synthesis from plasma low density lipoprotein (LDL). When grown in medium devoid of lipoproteins, the cells developed a minimal enhancement in steroid secretion in response to ACTH or cholera toxin. However, when LDL was available, steroid secretion was stimulated 4- to 9-fold. To determine the mechanism for this effect, we used LDL in which the protein component was labeled with 125I and the cholesteryl ester component was labeled with [3H]cholesteryl linoleate. These studies demonstrated that the cells derived cholesterol from LDL by binding the lipoprotein at a high affinity receptor site, internalizing it, and hydrolyzing its cholesteryl esters within lysosomes. The resultant free choles-

I

N ALL animal species so far studied, the bulk of the cholesterol used for the synthesis of steroid hormones in the adrenal cortex originates from plasma (1-6). Recently, the mechanism by which the adrenal gland takes up plasma cholesterol has begun to be explored. The possibility of a specific mechanism for such uptake was raised by the finding that cultured human fibroblasts possess receptors that enable them to bind and take up low density lipoprotein (LDL), the major cholesterolcarrying lipoprotein in human plasma (reviewed in Refs. 7 and 8). Human fibroblasts as well as a variety of other cell types (8) obtain their cholesterol by binding LDL at the receptor site and then internalizing the intact lipoprotein by adsorptive endocytosis. The internalized lipoprotein is delivered to cellular lysosomes where its cholesteryl esters are hydrolyzed. The resultant free choles-

Received August 10, 1978. Address requests for reprints to: Dr. Joseph L. Goldstein, Department of Molecular Genetics, University of Texas Health Science Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235. * This work was supported by USPHS Grant PO-l-HL-20948 from the NIH. f Recipient of fellowship grants from The Council for International Exchange of Scholars* and from the Paavo Nurmi Foundation, Helsinki, Finland.

terol was used for steroid synthesis and also acted to suppress the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol synthesis within the cell. LDL receptor activity was enhanced several-fold by treatment of the cells with ACTH or cholera toxin. High density lipoprotein, which did not bind to the LDL receptor, was not degraded with high affinity by the cells and did not support steroid synthesis. The current data suggest that the bovine adrenal cortex can obtain cholesterol for steroid hormone secretion from circulating LDL by means of a high affinity LDL receptor pathway. In a subsequent paper in this series, a similar high affinity LDL-binding site is demonstrated in membranes prepared from fresh bovine adrenocortical tissue. (Endocrinology 104: 599, 1979)

terol then becomes available to be used by the cell for membrane synthesis and regulatory purposes (7, 8). The relevance of this uptake system to adrenal physiology was recently shown by Faust et al., who studied the source of cholesterol for steroid hormone synthesis in the Y-l clone of cultured mouse adrenal cells (9). These cells, which were derived from a functional mouse adrenal tumor (10), were found to possess LDL receptors. Moreover, LDL taken up through the receptor mechanism served as the major source of cholesterol for steroid synthesis (9). High density lipoprotein (HDL), which was not recognized by the LDL receptor, did not stimulate steroid hormone synthesis in these mouse cells (9). Andersen and Dietschy (6, 11) and Balasubramaniam et al (5, 12) have used the drug 4-aminopyrazolopyrimidine as a tool to probe the source of adrenal cholesterol in intact rats. 4-Aminopyrazolopyrimidine blocks lipoprotein secretion from the liver in rats and, therefore, causes lipoproteins to fall to low levels in plasma. Both groups of workers found that when the plasma cholesterol level was reduced with 4-aminopyrazolopyrimidine in rats, the rate of cholesterol synthesis in the adrenal gland increased by 40- to 200-fold. This increase was 599

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mediated by a coordinate increase in the activities of two early enzymes in the cholesterol biosynthetic pathway, 3-hydroxy-3-methylglutaryl coenzyme A (CoA) synthase and 3-hydroxy-3-methylglutaryl CoA reductase (HMG CoA reductase) (5, 12). The rise in the level of these two enzymes in the adrenal gland was preceded by a dramatic fall in the adrenal content of cholesteryl esters after 4aminopyrazolopyrimidine treatment (5). Subsequent iv infusion of either human or rat lipoproteins restored the content of cholesteryl esters in the adrenal gland and caused a prompt suppression of HMG CoA synthase, HMG CoA reductase, and cholesterol synthesis (5, 6,12). This in vivo rat model system differed in one important respect from the mouse adrenal Y-l culture system. In the mouse Y-l cells, the lipoprotein receptor was specific for LDL (9), whereas in the 4-aminopyrazolopyrimidinetreated rat, HDL was more potent than LDL in causing an accumulation of cholesteryl esters and in suppressing cholesterol synthesis (5, 6, 11). Moreover, in studies of rat adrenal slices in vitro, Gwynne et al. found that the uptake of radiolabeled unesterified cholesterol was more rapid when the sterol was in HDL than when it was in LDL (13). The above studies raised the possibility that the rat adrenal might possess mechanisms that allow it to use cholesterol contained in either HDL or LDL, whereas the cultured mouse adrenal cells utilize a receptor that recognizes only LDL. The current studies were designed to assess the source of adrenal cholesterol in a system that allowed comparison of results in cultured cells and in membranes prepared from fresh tissue. These studies were made possible by the important work of Gospodarowicz, Hornsby, and Gill and their coworkers, who have succeeded in establishing proliferating monolayer cultures of bovine adrenocortical cells that are obtained by collagenase treatment of normal adult adrenal cortex (14-16). When these cells are grown in the presence of fibroblast growth factor, they can be subcultured after dissociation with trypsin and can be serially transferred throughout their life span of 55-65 generations (15). Hornsby and Gill have demonstrated that their method yields pure cultures of functioning adrenocortical cells (15, 16). These cells retain the ability to respond to ACTH, cholera toxin, and prostaglandin with an increase in steroid hormone production (14-16). The cultured cells differ from native adrenocortical cells, however, in that they rapidly lose the 11/?hydroxylase enzyme (Simonian, M., P. Hornsby, and G. Gill, personal communication). As a result, instead of producing cortisol and corticosterone, the normal products of bovine adrenal cortex, the cultured cells produce a variety of 11-deoxysteroids, including 11-deoxycortisol,1 1

Endo • 1979 Vol 104 • No 3

KOVANEN ET AL.

The trivial names used are: 11-deoxycortisol, 4-pregnen-17a,21-diol3,20-dione; 17a-hydroxyprogesterone, 4-pregnen-17a-ol-3,20-dione; 20adihydroprogesterone, 4-pregnen-20a-ol-3-one; 17a-hydroxy-20a-dihydroprogesterone, 4-pregnen- 17a,20a-diol-3-one.

progesterone, 17a-hydroxyprogesterone, 20a-dihydroprogesterone, and 17a-hydroxy-20a-dihydroprogesterone (Simonian, M., P. Hornsby, and G. Gill, personal communication). In order to correlate the behavior of adrenal cells cultured in vitro with the behavior of the adrenal gland in vivo, we obtained a line of cultured bovine adrenocortical cells from Hornsby and Gill and studied their lipoprotein and steroid metabolism. We found that the cells possess specific LDL receptors that are similar to the receptors previously found in the mouse Y-l cells. These LDL receptors supply cholesterol to the cells for use in steroid hormone formation. In the second paper of this series (17), we demonstrate that homogenates of fresh bovine adrenocortical tissue contain an LDL-binding site with properties that are similar to the functional receptor that could be demonstrated in the cultured bovine adrenocortical cells. Materials and Methods Materials [125I]NaI (11-17 mCi/iUg) and [l,2-3H]cholesterol (43 Ci/ mmol) were obtained from Amersham/Searle. DL-3-Hydroxy3-methyl[3-14C]glutaryl CoA (49.5 mCi/mmol), [2-uC]acetic acid, sodium salt (54 mCi/mmol), and ll-[4-uC]deoxycortisol (50 mCi/mmol) were purchased from New England Nuclear Corp. All steroids were obtained from Steraloids, Inc., except cholesterol which was purchased from Applied Sciences. Chloroquine diphosphate was purchased from Sigma Chemical Co. Aminogluthemide was provided by Ciba. ACTH was obtained as Acthar from Armour Pharmaceutical Co. Purified cholera toxin was kindly provided by Dr. Richard Finkelstein. Purified fibroblast growth factor was purchased from Collaborative Research, Inc. (Waltham, MA). Powdered F-12 (Ham) medium (catalog no. 430-1700) was obtained from Grand Island Biological Co. The reconstituted F-12 medium was stored at —20 C, and thawed and used on the same day. Fetal bovine serum was obtained from Irvine Scientific Sales Co., Inc. (catalog no. 300, lot 707201), or Flow Laboratories (catalog no. 29-101-49, lot 40551322). Petri dishes were obtained from Falcon Plastics. Other tissue culture supplies, thin layer and gas-liquid chromatographic materials, and reagents for assays were obtained from sources as previously reported (9, 18). Cultured cells A vial of frozen primary adult bovine adrenocortical cells (15) was kindly provided by Drs. Peter J. Hornsby and Gordon N. Gill. Cells were grown in monolayer, subcultured as described by Hornsby and Gill (15), and used for experiments between the 5th and 12th passage (10th to 25th generation). Stock cultures of cells were maintained in a humidified incubator (5% CO2) at 37 C in Falcon petri dishes (100 X 20 mm) containing 7 ml growth medium A [Ham's F-12 medium supplemented with penicillin (100 U/ml), streptomycin (100 jug/ml), and 14 mM NaHCO3] containing 40 ng/ml fibroblast growth factor and 10% (vol/vol) fetal bovine serum. All exper-


LDL RECEPTORS IN BOVINE ADRENAL CELLS iments were carried out using a similar format. Confluent monolayers of cells from stock cultures were dissociated by incubation for 1 min at 37 C in a solution containing 0.05% trypsin, 0.02% EDTA, 0.14 M NaCl, 5 mM KC1, 10 mM NaHCO,,, and 1% sucrose according to the procedure of Hornsby and Gill (15). Cells were seeded (day 0) at a concentration of 1 x 105 cells/dish into Falcon petri dishes (60 x 15 mm) containing 3 ml medium A with. 20 ng/ml fibroblast growth factor and 10% fetal bovine serum. On days 3 and 5, the medium was replaced with 3 ml fresh medium A containing 20 ng/ml fibroblast growth factor and 10% fetal bovine serum. On day 6, each cell monolayer was washed with 3 ml phosphate-buffered saline, after which 2 ml medium A containing 10% bovine lipoproteindeficient serum and the indicated concentration of ACTH or cholera toxin were added. Experiments were initiated on day 7 or 8, as indicated. Lipoproteins Human and bovine LDL (density, 1.019-1.063 g/ml), human HDL (density, 1.090-1.215), bovine HDL (density, 1.085-1.215), and human and fetal bovine lipoprotein-deficient serum (density, >1.215) were fractionated by differential ultracentrifugation, as previously described (18, 19). Human lipoprotein fractions were prepared from single 500-ml units of blood collected in 0.1% EDTA from individual healthy subjects (18). Bovine blood was obtained from a local slaughterhouse, and the lipoprotein fractions were prepared from single 1-liter units of blood collected in 0.1% EDTA from individual cows or steers. Both the human and bovine plasma were processed within 24 h of bleeding. Each lipoprotein fraction migrated as a homogeneous band on lipoprotein electrophoresis (18). For human LDL and HDL, the mass ratios of total cholesterol to protein were 1.5:1 and 1:3, respectively. For bovine LDL and HDL, the mass ratios of total cholesterol to protein content were 1:1 and 1:1.7, respectively. The concentrations of LDL and HDL are expressed in terms of their protein content. Lipoproteins were radiolabeled with 125I, as previously described (20, 21). The specific activities were: human [125I]iodo-LDL, 200-400 cpm/ng protein; bovine [125I]iodo-LDL, 530-1170 cpm/ng; human [125I]iodoHDL, 350 cpm/ng; and bovine [125I]iodo-HDL, 840 cpm/ng. In all preparations, more than 98% of the radioactivity was precipitated by incubation with 15% trichloroacetic acid, and less than 2% was extractable into chloroform-methanol. For experiments, each [125I]iodolipoprotein was diluted with the corresponding unlabeled lipoprotein to give the final concentration and specific activity indicated. LDL radiolabeled with [:'H]cholesteryl linoleate (r-[CL;| H]LDL) was prepared by the reconstitution method of Krieger et al. in which the endogenous cholesterol and cholesteryl esters of LDL are removed by heptane extraction and replaced with exogenous [;1H]cholesteryl linoleate (22). Assays The activity of HMG CoA reductase was measured by determining the rate of conversion of [14C]HMG CoA (12,000 cpm/nmol) to [14C]mevalonate in detergent-solubilized cell extracts (18). The total cellular content of [125I]iodolipoproteins (surface bound plus internalized) and the rate of their proteo-

601

lytic degradation by intact monolayers were determined as previously described (21). When [125I]iodo-LDL was used, the amount bound to the cell surface and the amount internalized by the cells were measured separately by the dextran sulfate release assay (23). The incorporation of [uC]acetate into [14C]cholesterol by intact monolayers was measured by a previously described method in which the cells were saponified with ethanolic KOH, the 14C-labeled nonsaponifiable lipids were extracted with petroleum ether, and the [uC]cholesterol was isolated and quantified by thin layer chromatography (24). The protein concentration of cell extracts, whole cells, and lipoproteins was determined by the method of Lowry et al. (25) with bovine serum albumin as a standard. Measurement of steroids The cellular content of unesterified and esterified cholesterol was determined by a previously described method in which the steroids were extracted from washed cell pellets with chloroform-methanol (2:1), the unesterified and esterified cholesterol fractions were separated on silicic acid-Celite columns, and, after alkaline hydrolysis of the cholesteryl ester fraction, the cholesterol content of each fraction was measured by gas-liquid chromatography (26). The amount of steroids secreted by the adrenocortical cells was estimated by measuring the content of fluorogenic steroids in the culture medium (16). The medium (2 ml) from one petri dish of cultured cells was agitated on a Vortex mixer for 10 sec in a test tube containing 7 ml methylene chloride. The phases were separated by centrifugation (1500 rpm for 15 min at 24 C). The upper (aqueous) phase was discarded and the lower (methylene chloride) phase was washed with 2 ml 0.1 N NaOH. The final bottom phase was reacted with 2 ml ice-cold 65% sulfuric acid in ethanol. After 30 min at room temperature, the upper (methylene chloride) phase was discarded and the lower (ethanolic surfuric acid) phase was used for determination of fluorescence intensity, with an excitation wavelength of 470 nm and an emission wavelength of 520 nm, in an Aminco Bowman fluorometer (27). To standardize these measurements, in each experiment the observed fluorescence was related to a standard curve generated by the addition of known amounts of cortisol to aliquots of incubation medium that were subsequently extracted and assayed for fluorescence, as described above. The results represent the micrograms of fluorogenic cortisol equivalents per ml culture medium and are expressed as micrograms of steroid per dish or per mg cell protein. For each experiment, a blank extraction was conducted using medium A containing 10% bovine lipoprotein-deficient serum that had not been incubated in the presence of cells. Hydrolysis ofr-[CL-3HJLDL and incorporation of [3H]'cholesterol into 3H-labeled steroids by intact adrenocortical cells Monolayers were incubated with r-[CL-3H]LDL in 2 ml medium for 48 h at 37 C, after which the medium was removed and pooled, as described in the legend to Table 5. The secreted steroids were extracted, as described above for measurement of fluorogenic steroids, except that ll-[14C]deoxycortisol (9000 cpm) was added to each sample as a recovery standard. The evaporated organic extract was dissolved in 40 /xl chloroform-


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602

methanol (2:1) and spotted on plastic-backed silica gel (without gypsum) thin layer sheets. The sheets were developed with benzene-ethanol (12:1). The steroids were visualized by iodine vapor with the following Rf values: 17a-hydroxy-20a-dihydroprogesterone, 0.13; 11-deoxycortisol, 0.16; and 17a-hydroxyprogesterone, 0.27. The unresolved band corresponding to the first two steroids (Rf = 0.13-0.16) and the band containing the third steroid (Rf = 0.27) were separately scraped from the thin layer sheet and eluted from the silica gel with three extractions of 2.5 ml chloroform-methanol (2:1, vol/vol). (The Rf value for progesterone in this system was 0.42). The two extracts were then subjected to reverse phase high pressure liquid chromatography using a jiiBondapak Cis column (length, 30 cm; internal diameter, 3.9 mm) with a water-methanol (60:40, vol/vol) solvent system. The flow rate was 2 ml/min and the pressure was 2500 p.s.i. Fractions of 1 ml each were collected and analyzed for their content of 3H radioactivity. The retention times for 11deoxycortisol, 17a-hydroxy-20a-dihydroprogesterone, and 17ahydroxyprogesterone were 5.2, 8.1, and 8.2 min, respectively. Thus, the two-step thin layer and high pressure liquid chromatography procedure allowed unambiguous measurements of each of these three steroids. After the medium was removed, the amount of unesterified [3H]cholesterol formed from the hydrolysis of r-[CL-3H]LDL and contained within the cells was determined as previously described (22). Results are expressed as the nanomoles of 3Hlabeled steroids formed from r-[CL-3H]LDL per mg of total cell protein.

Results To measure the effects of lipoproteins on steroid secretion by the cultured bovine adrenocortical cells, we employed a standard assay in which the cells were grown to near confluence in the presence of fetal bovine serum supplemented with fibroblast growth factor (14-16). Cells were then switched to medium containing bovine lipoprotein-deficient serum supplemented with measured amounts of human or bovine lipoproteins as indicated. Steroid secretion was stimulated by the addition of cholera toxin or ACTH. As originally determined by Simonian, Hornsby, and Gill (personal communication) and as confirmed below, the bovine adrenocortical cells secrete a mixture of fluorogenic and nonfluorogenic steroids in response to ACTH or cholera toxin. As a routine index of steroid output, we measured the content of fluorogenic steroids in the medium and related the observed fluorescence to the measured fluorescence of a known standard of cortisol (15, 16). The results are expressed as micrograms of steroid per dish or per mg cell protein. Although cortisol is not one of the major fluorogenic steroid products of these cells,2 this steroid was used as the standard because it permitted a reproducible comparison of the 2 As recently determined by M. Simonian, P. Hornsby, and G. Gill, the two principal fluorogenic steroids secreted by the bovine adrenocortical cells are 20a-dihydroprogesterone and its 17a-hydroxy derivative (personal communication).

Endo 1979 Vol 104 . No 3

relative response of these cells to various stimuli (15,16). The data in Fig. 1 show that when the cells were grown as described above and incubated in lipoprotein-deficient serum, a small amount of fluorogenic steroids was secreted over 72 h. In the absence of a stimulus to steroid secretion, the addition of either LDL or HDL caused no appreciable increase in the content of fluorogenic steroids in the medium (Fig. 1A). The addition of cholera toxin caused a detectable but minor stimulation of fluorogenic steroid secretion when the medium contained no lipoproteins (Fig. IB). However, when human LDL was present, a marked stimulation of fluorogenic steroid secretion occurred in response to cholera toxin. In contrast, human HDL did not enhance the secretion of fluorogenic steroids by these cells (Fig. IB). In other experiments, we showed that bovine LDL behaved similarly to human LDL. When added to the medium at a concentration of 50 jiig protein/ml in the presence of cholera toxin, bovine LDL produced a 9-fold stimulation in fluorogenic steroid secretion, whereas bovine HDL (100 jug protein/ml) had no effect. Figure 2 compares the response of the bovine adrenocortical cells to increasing concentrations of ACTH or cholera toxin in the absence and presence of human LDL. Maximal secretion of fluorogenic steroids was achieved at an ACTH concentration of 1 mU/ml. The attainment of this response required the presence of LDL in the A. No Cholera Toxin

B. Cholera Toxin

1.0 Additions to Medium E3 None • LDL • HDL

0.8

§ | ? 0-6 0.4 0.2

0

Oto24 Hours

24 to 48 48 to 72 Oto24 24 to 48 Hours Hours Hours Hours TIME OF INCUBATION

48to72 Hours

FIG. 1. Quantitation of fluorogenic steroids secreted by monolayers of bovine adrenocortical cells during successive 24-h intervals after addition of cholera toxin and human lipoproteins to the culture medium. On day 6 of cell growth, each monolayer received 2 ml medium A containing 10% bovine lipoprotein-deficient serum and either no cholera toxin (A) or 0.8 /ig/ml cholera toxin (B). On day 7 (zero time), the medium was replaced with 2 ml medium A containing 10% bovine lipoprotein-deficient serum, either no cholera toxin (A) or 0.8 /xg/ml cholera toxin (B), and one of the following human lipoproteins: none, 47 jug protein/ml LDL, or 96 jug protein/ml HDL. Every 24 h the medium was replaced with fresh medium containing the indicated addition. At the indicated interval, the medium from duplicate dishes was removed for measurement of its fluorogenic steroid content. The average content of total cell protein in each dish at the end of the experiment at 72 h was 300 ng.


LDL RECEPTORS IN BOVINE ADRENAL CELLS .. B. Cholera Toxin

ACTH

4.8 4.0

\

3.2 2.4 16 0.8 0

0 10"6 1O'5 10' 4 1O"3 1O"2 1O"1 ACTH (U/ml)

0

10"4 10' 3 10"2 10"1

603

[125I]iodo-LDL, about 10% of the total cell-bound lipoprotein was releasable by dextran sulfate (Fig. 3A, inset). These data are similar to those previously observed in human fibroblasts in which about 10-15% of the cellbound [125I]iodo-LDL in the steady state at 37 C is located on the cell surface (23). In addition to the high affinity uptake process for [125I]iodo-LDL, which showed saturation at 50-100 jtig protein/ml, the cells exhibited a nonsaturable uptake process whose rate increased gradually in a linear fashion as the concentration of [125I]iodoLDL was increased in the culture medium above 50-100 jiig protein/ml (Fig. 3A). In contrast to the high affinity uptake process, which was observed for [125I]iodo-LDL

CHOLERA TOXIN (^g/ml)

FIG. 2. Stimulation of fluorogenic steroid secretion in monolayers of bovine adrenocortical cells by varying concentrations of ACTH (A) and cholera toxin (B) in the absence (O) and presence (•) of human LDL. On day 6 of cell growth, each monolayer received 2 ml medium A containing 10% bovine lipoprotein-deficient serum and the indicated concentration of either ACTH or cholera toxin. On days 7 and 8, the medium was replaced with 2 ml medium A containing 10% bovine lipoprotein-deficient serum, the indicated concentration of either ACTH or cholera toxin, and either no LDL (O) or 50 jug protein/ml LDL (•). On day 9, the medium was removed for measurement of the amount of fluorogenic steroids that had accumulated during the 24-h interval between days 8 and 9. Each value represents the average of duplicate incubations.

culture medium (Fig. 2A). The maximal response to cholera toxin was similar to that of ACTH and, again, the attainment of this response required the presence of LDL in the culture medium (Fig. 2B). The secretion of fluorogenic steroids was abolished in the presence of aminoglutethimide at a concentration of 100 /i.g/ml (data not shown). The above data suggest that the bovine adrenocortical cells possess LDL receptors that allow them to utilize the cholesterol of LDL, but not of HDL, for steroid hormone synthesis. To study the uptake of lipoproteins directly, we compared the uptake and degradation of human [125I]iodo-LDL and [125I]iodo-HDL in bovine adrenocortical cells incubated at 37 C. The data in Fig. 3 show that as the content of [125I]iodo-LDL in the culture medium was increased, the total cellular content of 125I radioactivity rose with saturation kinetics (Fig. 3A). As in other cultured cell systems, the [125I]iodo-LDL that was bound to the receptor at the cell surface could be released by treatment of the cells with dextran sulfate (23). Figure 3A (inset) shows the amount of dextran sulfate-releasable [125I]iodo-LDL at; each concentration of lipoprotein. Maximal receptor binding occurred at an LDL concentration of 50-100 jug protein/ml, with half-maximal binding occurring at about 15 jug protein/ml. This affinity is similar to the affinity of the human fibroblast LDL receptor (8). After incubation for 5 h at saturating concentrations of

50

100

150

200

125

I-LIP0PR0TEIN (^g protein/ml)

FIG. 3. Saturation kinetics for total uptake (A) and degradation (B) of human [l2r'I]iodo-LDL (A) and human [125I]iodo-HDL (•) in monolayers of bovine adrenocortical cells. On day 6 of cell growth, each monolayer received 2 ml medium A containing 10% bovine lipoproteindeficient serum and 0.4 /xg/ml cholera toxin. On day 8, the medium was replaced with 2 ml medium A containing 10% human lipoproteindeficient serum, 0.4 /xg/ml cholera toxin, and the indicated concentration of either [l25I]iodo-LDL (87 cpm/ng) or [12flI]iodo-HDL (64 cpm/ ng). After incubation for 5 h at 37 C, the total cellular content of [l2r'I]iodolipoprotein (A) and the total amount of [''2r>I]iodolipoprotein degraded (B) were determined. The inset A (inset) shows the fraction of the total cellular content of [12r>I]iodo-LDL bound to the cell surface receptor, as determined by the dextran sulfate release assay (23). Each value represents a single incubation.


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but not for [125I]iodo-HDL, the nonsaturable uptake process was similar for both LDL and HDL (Fig. 3A). Thus, the slope of the [125I]iodolipoprotein uptake curve at [125I]iodo-LDL concentrations above 50 /i.g/ml was similar to the slope for [125I]iodo-HDL at all concentrations. Figure 3B demonstrates that the bovine adrenocortical cells degraded the [125I]iodo-LDL at a rate that was proportional to the amount of receptor binding and uptake, with saturation achieved at a concentration of 50-100 ^g protein/ml. At concentrations of LDL below saturation, approximately 2.5 times as much [12oI]iodoLDL had been degraded by the cells in 5 h (Fig. 3B) as was contained within the cell in the steady state (Fig. 3A). This relation between the steady state concentration of LDL within the cell and the rate of degradation was similar to that seen previously in human fibroblasts (23). [125I]Iodo-HDL, which was not taken up by a high affinity process, was not degraded with high affinity by the bovine adrenocortical cells (Fig. 3B). The specificity of the LDL receptor was tested by comparing the ability of unlabeled human LDL and HDL to compete with human [125I]iodo-LDL for surface binding and degradation in the bovine adrenocortical cells. As shown in Fig. 4, unlabeled LDL was considerably more effective than HDL in competing with [125I]iodoLDL for both surface binding and degradation. The preceding experiments were all performed with 125 I-labeled human lipoproteins. The data in Fig. 5 demonstrate that the bovine adrenocortical cells were able to B. Degradation

A. Surface Binding

100

200

300

400

0

100

200

300

400

UNLABELED LIPOPROTEIN (Mg protein/ml)

FIG. 4. Comparison of the ability of human LDL (A) and human HDL (•) to compete with human [125I]iodo-LDL for the surface binding (A) and degradation (B) in monolayers of bovine adrenocortical cells at 37 C. On day 6 of cell growth, each monolayer received 2 ml medium A containing 10% bovine lipoprotein-deficient serum and 0.1 U/ml ACTH. On day 7, each monolayer received 2 ml medium A containing 10% human lipoprotein-deficient serum, 0.1 U/ml ACTH, 10 jug protein/ml [125I]iodo-LDL (125 cpm/ng), and the indicated concentration of either unlabeled LDL (A) or HDL (•). After incubation for 5 h at 37 C, the amounts of dextran sulfate-releasable [125I]iodo-LDL (surface binding) and [125I]iodo-LDL degraded were determined, as described in Materials and Methods. Each value represents a single incubation, except for those dishes that received no unlabeled lipoprotein (•) which represent the mean of triplicate incubations.

< LU Q

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1979 No 3

800 600 -

LU

400

-

o a. 200

' I - L I P O P R O T E I N ^ g protein/ml)

FIG. 5. Saturation kinetics for degradation of bovine [l25I]iodo-LDL (•) and bovine [125I]iodo-HDL (A) in monolayers of bovine adrenocortical cells. On day 6 of cell growth, each monolayer received 2 ml medium A containing 10% bovine lipoprotein-deficient serum and 0.8 jug/ml cholera toxin. On day 8, the medium was replaced with 2 ml medium A containing 10% human lipoprotein-deficient serum, 0.8 fig/ml cholera toxin, and the indicated concentration of either [12r>I]iodo-LDL (37 cpm/ng) or [125I]iodo-HDL (50 cpm/ng). After incubation for 5 h at 37 C, the total amount of [125I]iodolipoprotein degraded was determined. Each value represents a single incubation.

degrade bovine [125I]iodo-LDL with saturation kinetics that were similar to those for human [125I]iodo-LDL. Moreover, these cells did not show a high affinity degradation process for bovine [125I]iodo-HDL. The affinity of the cells for bovine [125I]iodo-LDL was slightly higher than their affinity for human [125I]iodo-LDL (half-maximal degradation at 7 jug protein/ml for the bovine [125I]iodo-LDL and 15 jiig/ml for the human [125I]iodoLDL). However, the maximal rate of degradation of human [125I]iodo-LDL was 2-fold greater on the average than the maximal rate of degradation of bovine [125I]< iodo-LDL in several studies in which the two lipoproteins were compared directly. As previously observed in other types of cultured cells (8), the uptake of bovine LDL through the receptor system was associated with a suppression of HMG CoA reductase activity in the bovine adrenocortical cells (Table 1). On the other hand, bovine HDL, which was not taken up or degraded with high affinity, did not suppress HMG CoA reductase activity. The addition of ACTH did not stimulate HMG CoA reductase activity when the cells were incubated in the absence of LDL, nor did it affect the ability of LDL to suppress the enzyme (Table 1). In other experiments not shown, incubation of the bovine adrenocortical cells with 25 ju,g protein/ml human LDL for 16 h caused a 95% suppression of HMG CoA reductase activity, whereas human HDL caused no significant suppression at concentrations as high as 475 jug protein/ml. In cultured human fibroblasts and mouse Y-l adrenal cells, the suppression of HMG CoA reductase by LDL


LDL RECEPTORS IN BOVINE ADRENAL CELLS requires hydrolysis of the cholesteryl esters of the lipoprotein within lysosomes and, hence, this suppression can be blocked by the lysosomal enzyme inhibitor chloroquine (9, 28). The data in Table 2 show that a similar requirement was present in the bovine adrenocortical cells. Thus, human LDL suppressed HMG CoA reductase when added to the cells, and this suppression was prevented by chloroquine. As a control for the chloroquine effect, we measured the ability of 7-ketocholesterol to suppress HMG CoA reductase. In fibroblasts and mouse Y-l adrenal cells, this polar sterol, when added in a nonlipoprotein form, suppresses HMG CoA reductase without a requirement for lysosomal action (9, 28). The data in Table 2 show that 7-ketocholesterol suppressed HMG CoA reductase activity also in the bovine adrenocortical cells, and its effect was not inhibited by chloroquine. TABLE 1. Comparison of the ability of bovine LDL and HDL to suppress HMG CoA reductase activity in bovine adrenocortical cells

Addition to medium

Concentration of lipoprotein in medium (/ig protein/ml)

None LDL LDL HDL HDL

0 21 42 55 97

HMG CoA reductase activity (pmol-min"1 mg protein"1) - ACTH

+ ACTH

168 24 12 188 194

132 21 15 167 174

On day 6 of cell growth, each monolayer received 2 ml medium A containing 10% bovine lipoprotein-deficient serum and either no ACTH or 0.1 U/ml ACTH, as indicated. On day 7, the medium was replaced with 2 ml medium A containing 10% bovine lipoprotein-deficient serum, either no ACTH or 0.1 U/ml ACTH (as indicated), and the indicated concentration of either bovine LDL or bovine HDL. After incubation for 24 h at 37 C, the cells were harvested for measurement of HMG CoA reductase activity. Each value represents the average of duplicate incubations. TABLE 2. Prevention by chloroquine of LDL-mediated suppression of HMG CoA reductase activity in bovine adrenocortical cells

Addition to medium

None Human LDL (21 ng protein/ml) 7-Ketocholesterol (2 /ig/ml)

HMG CoA reductase activity (pmol-min"1 mg protein"1) - Chloroquine

-I- Chloroquine

120 26 7.3

225 220 8.7

On day 6 of cell growth, each monolayer received 2 ml medium A containing 10% bovine lipoprotein-deficient serum and 0.5 jug/ml cholera toxin. On day 8, the medium was replaced with 2 ml medium A containing 10% bovine lipoprotein-deficient serum, 0.5 jug/ml cholera toxin, 5 fil ethanol, and one of the indicated additions in the absence or presence of 75 JUM chloroquine. After incubation for 7 h at 37 C, the cells were harvested for measurement of HMG CoA reductase activity. Each value represents the average of triplicate incubations.

605

In human fibroblasts and mouse Y-l adrenal cells, the lysosomal hydrolysis of the cholesteryl esters of LDL enhances the rate at which the cells incorporate [14C]oleate into cholesteryl [14C]oleate (9, 29). This is due to an activation of a microsomal acyl-CoA-cholesterol acyltransferase by excess cholesterol derived from LDL (8). In experiments not shown, we demonstrated that human LDL, but not HDL, markedly increased the rate at which the bovine adrenocortical cells incorporated [14C]oleate into cholesteryl [14C]oleate. In contrast to its lack of effect on HMG CoA reductase activity (Table 1), ACTH caused a 2-fold enhancement in the activity of the LDL receptor when the hormone was added to the cells in the absence of lipoproteins (Table 3). Cholera toxin produced a somewhat greater enhancement in receptor activity. When the cells were subjected to prior incubation with LDL in the absence of ACTH or cholera toxin, the activity of the LDL receptor was reduced by 80%, a finding similar to that observed in human fibroblasts (30). However, even in the presence of LDL, the addition of ACTH or cholera toxin caused a 1.5- to 2-fold increase in LDL receptor activity, as measured by the binding, internalization, and degradation assays (Table 3). TABLE 3. Regulation of LDL receptor activity in bovine adrenocortical cells by prior incubation with human LDL and ACTH or cholera toxin LDL receptor activity Addition to medium during 24 h prior incubation

None ACTH (0.1 U/ml) Cholera toxin (0.8 /ig/ml) LDL (50 /ig protein/ml) LDL + ACTH LDL + cholera toxin

[12BI]Iodol2S [12r'lllodoLDL bound [ I]IodoLDL interLDL deto cell surnalized graded (ng-5 face (ng/mg h"1 mg pro(ng/mg protein) tein"1) protein) 35, 29 68,62 100, 95 5, 4 10, 5 9, 4

249, 241 517,506 744, 726 58, 44 94, 92 72, 66

1200, 1130 2590,2830 4760, 4660 266, 229 502, 474 479, 405

On day 6 of cell growth, each monolayer received 2 ml medium A containing 10% bovine lipoprotein-deficient serum and the indicated addition. After incubation for 24 h at 37 C, each monolayer was washed twice with 3 ml phosphate-buffered saline containing 10% bovine lipoprotein-deficient serum, after which were added 2 ml medium A containing 10% human lipoprotein-deficient serum and 10 jug protein/ml human [125I]iodo-LDL (112 cpm/ng) in the absence or presence of 500 /tg protein/ml unlabeled human LDL. After incubation for 5 h at 37 C, the medium was removed and its content of l25I-labeled trichloroacetic acid-soluble material was determined. The cell monolayers were then washed by the standard technique (23), and the amounts of cell surfacebound and internalized [12)I]iodo-LDL were determined by the dextran sulfate release assay (23). All data shown represent values for the high affinity processes, which were calculated by subtracting the nanograms of [125I]iodo-LDL bound, internalized, and degraded in the presence of the excess unlabeled LDL from the nanograms of [l25I]iodo-LDL bound, internalized, and degraded in the absence of the unlabeled LDL (21). Both values for each duplicate incubation are shown.


KOVANEN ET AL.

606

Overall, the addition of LDL to the culture medium of the bovine adrenocortical cells resulted in an increase in the cellular content of unesterified and esterified cholesterol (Table 4). This increase was similar in the absence and presence of ACTH. In contrast, the addition of HDL did not affect the cellular content of unesterified or esterified cholesterol either in the absence or presence of ACTH. In the same experiment, LDL caused a severalfold enhancement in the secretion of fluorogenic steroids in the medium, whereas HDL caused no such enhancement. It is of interest that the increase in the total cholesterol content in the cells (unesterified plus esterified cholesterol) in the presence of ACTH (a total of 30 /xg steroid/mg protein over 24 h in the presence of 100 /xg/ml LDL) was much greater than the increase in fluorogenic steroid output (1.25 /xg steroid/mg protein). Since the measurement of fluorogenic steroids represents

a significant underestimate of the total steroid output by these cells (Simonian, M., P. Hornsby, and G. Gill, personal communication), it is not possible to compare directly the amount of LDL-cholesterol used for enrichment of cellular cholesterol stores with that used for conversion to steroid hormones. However, it seems likely that when LDL is added to cultured adrenocortical cells that have been deprived of lipoproteins in either the presence or absence of ACTH, most of the exogenous cholesterol obtained from LDL over the initial 24 h is used to expand the cellular cholesterol stores and only a relatively small fraction is converted to secreted steroids. Inasmuch as many of the steroids secreted by the bovine adrenocortical cells are not fluorogenic (Simonian, M., P. Hornsby, and G. Gill, personal communication), we performed an experiment to determine directly whether the cholesteryl esters of LDL could serve as precursors for other steroid hormones. To perform this experiment, we extracted LDL with heptane and then reconstituted the core of the lipoprotein with exogenous [3H]cholesteryl linoleate (22). The reconstituted particle,

Endo Vol 104

1979 No 3

designated r-[CL-3H]LDL, has been shown previously to bind to the LDL receptor and to be taken up and hydrolyzed in human fibroblasts, supplying cholesterol to these cells (22). The r-[CL-3H]LDL is not hydrolyzed at high rates by cells that lack LDL receptors (22). The data in Table 5 show that when the bovine adrenocortical cells were incubated with r-[CL-3H]LDL in the absence of a stimulus to steroid hormone secretion, the [3H] cholesteryl linoleate was hydrolyzed, and unesterified [3H]cholesterol accumulated in the cells. Under these conditions, only a relatively small amount of 3H-labeled steroids appeared in the culture medium. On the other hand, when the incubation was performed in the presence of TABLE 5. Hydrolysis of r-[CL-3H]LDL and secretion of 3H-labeled steroids by bovine adrenocortical cells incubated in the absence and presence of cholera toxin Conversion of r-[CL-3H]LDL to (nmol formed/mg protein) Addition to medium

None Cholera toxin

Unesterified [3H]cholesterol in cells

17a-[3H]Hydroxyprogesterone in medium

18.3 22.0

0.05 0.34

Deoxycortisol in medium 0.09 1.18

17a-[3H]Hydroxy20a-dihydroprogesterone in medium 0.02 0.11

On day 6 of cell growth, each of 30 monolayers received 2 ml medium A containing 10% bovine lipoprotein-deficient serum and either no cholera toxin (15 monolayers) or 0.1 jug/ml cholera toxin (15 monolayers), as indicated. On day 7, the medium was replaced with 2 ml medium A containing 10% bovine lipoprotein-deficient serum, either no cholera toxin or 0.1 jug/ml cholera toxin (as indicated), and 9 /xg protein/ml r-[CL-3H]LDL (120,000 cpm/nmol cholesteryl linoleate). After incubation at 37 C for 48 h, the medium from each set of 15 dishes was pooled and the content of 3H-labeled steroids in the medium was measured, as described in Materials and Methods. The monolayers were then washed and harvested and the cellular content of unesterified [3H]cholesterol was measured. Each value represents the average of duplicate measurements.

TABLE 4. Increase in cellular content of cholesterol and stimulation of fluorogenic steroid formation by human LDL, but not HDL, in bovine adrenocortical cells Lipoprotein concentration in Lipoprotein addition to medium medium (jug protein/ml) None LDL LDL HDL HDL

0 10 100 30 300

Unesterified cholesterol in cells (jug sterol/mg protein)

lated 15 24 30 15 16

+ ACTH 13 24 32 16 15

Esterified cholesterol in cells (fig sterol/mg protein)

Fluorogenic steroids in medium (/xg steroid/mg protein)

Unstimulated

+ ACTH

Unstimulated

+ ACTH

1.3 4.1 12 1.1 1.1

0.34 0.70 0.67 0.34 0.41

0.75

0.91 3.7 10 1.8 1.2

1.8 2.0 0.80

0.79

On day 6 of cell growth, each monolayer received 2 ml medium A containing 10% bovine lipoprotein-deficient serum and 0.1 U/ml ACTH as indicated. On day 8, the medium was replaced with 2 ml medium A containing 10% bovine lipoprotein-deficient serum, 0.1 U/ml ACTH (as indicated), and the indicated concentration of either human LDL or HDL. After incubation for 24 h at 37 C, the medium was removed for measurement of its content of fluorogenic steroids and the cells were harvested for measurement of their content of unesterified and esterified cholesterol. Each value represents the average of duplicate incubations.


LDL RECEPTORS IN BOVINE ADRENAL CELLS TABLE 6. Suppression of cholesterol synthesis and stimulation of fluorogenic steroid secretion by human LDL in bovine adrenocortical cells

Addition to medium

None Cholera toxin (0.8 jtg/ml) LDL (50 ng protein/ml) Cholera toxin + LDL

[14C]Acetate incorporation into [14C]cholesterol (cpm X l(T7mg protein) 109 107 8.1 2.0

FluoroTotal chogenic stelesterol in roids in mecells (ng dium (jLtg sterol/mg steroid/mg protein) protein) 9.6 9.6 15 17

0.55 0.96 0.59 4.6

On day 5 of cell growth, each monolayer received 2 ml medium containing 10% bovine lipoprotein-deficient serum, 40 ng/ml fibroblast growth factor, and either no cholera toxin or cholera toxin, as indicated. On day 6, the medium was replaced with 2 ml medium A containing 10% bovine lipoprotein-deficient serum, either 1 mM unlabeled sodium acetate or 1 mM sodium [2-14C]acetate (54,000 cpm/nmol), and cholera toxin or LDL, as indicated. After incubation for 48 h at 37 C, the dishes were divided into two groups. In those dishes receiving unlabeled sodium acetate, the medium was removed for measurement of its content of fluorogenic steroids and the cells were harvested for measurement of their content of total cholesterol. In those dishes receiving sodium [2-14C]acetate, the medium and cells were harvested together for measurement of the incorporation of [14C]acetate into total [l4C]cholesterol. Each value represents the average of quadruplicate incubations.

cholera toxin, an increased amount of 3H-labeled steroids appeared in the medium during the 48-h incubation. Of the three steroids that were isolated, the largest amount of 3H radioactivity was found in 11-deoxycortisol (compound S). Cholera toxin produced a 13-fold increase in the amount of [3H]cholesterol incorporated into this steroid. This result is consistent with the recent data of Simonian et al. (personal communication), who showed that 11-deoxycortisol is one of the principal steroids secreted by these cultured bovine adrenocortical cells. The generation of unesterified [3H] cholesterol and 11[3H]deoxycortisol from r-[CL-3H]LDL were both shown to be inhibited by more than 95% when a 25-fold excess of unlabeled LDL was included in the culture medium, thus indicating that the formation of both compounds was mediated through the LDL receptor. To confirm that the LDL-mediated suppression of HMG CoA reductase was associated with a suppression of sterol synthesis in the bovine adrenocortical cells, we measured the effect of the lipoprotein on cholesterol synthesis from [l4C]acetate. The data in Table 6 demonstrate that LDL suppressed [14C]acetate incorporation into [14C]cholesterol by more than 90% in the absence or presence of cholera toxin. At the same time that it suppressed cholesterol synthesis, LDL produced an increase in the total cholesterol content of the cell. Moreover, in the presence of cholera toxin, LDL caused a 4.8fold increase in the amount of fluorogenic steroids se-

607

creted ini.o the medium. The fact that both the cellular cholesterol content and the amount of fluorogenic steroid output increased in the presence of LDL at a time when cholesterol synthesis was suppressed by more than 90% indicated that both the cellular cholesterol and the fluorogenic steroids were derived from LDL. Discussion The studies in this paper demonstrate that adrenocortical cells cultured from normal adult bovine adrenal glands by the method of Hornsby and Gill and their coworkers (14-16) possess LDL receptors. Moreover, the cells utilize these receptors to take up and degrade LDL and thereby obtain the cholesterol that they need for steroid hormone synthesis. When the cells are grown in the absence of lipoproteins, their ability to develop enhanced steroid secretion in response to ACTH or cholera toxin is limited. If LDL is added to the culture medium, the lipoprotein binds to the receptor, is taken up by the cells, and provides substrate for steroid hormone synthesis. As an index of steroid secretion in these studies, we routinely measured the content of fluorogenic steroids in the medium (14-16). LDL caused a 4- to 9-fold increase in the content of these steroids when added to cells that had been previously stimulated with ACTH or cholera toxin. By using LDL in which the cholesteryl esters were labeled with [3H]cholesteryl linoleate, we were able to show that the [3H]cholesterol released from the hydrolysis of LDL was converted to several side chain cleavage products, including 11-deoxycortisol which has been shown by Simonian et al. to be one of the major steroids secreted by these cells (personal communication). In addition to supplying cholesterol for steroid hormone synthesis, LDL also suppressed HMG CoA reductase activity in the adrenal cells. This response was similar to the response in other cell types in which LDL is taken up through the receptor mechanism (7-9). In the absence of LDL, the adrenal cells developed high HMG CoA reductase activity and high rates of cholesterol synthesis from [14C]acetate, but the cells were unable to support a maximal rate of steroid hormone synthesis. These data indicate that the bovine adrenocortical cells are able to develop rates of cholesterol synthesis that are sufficient to support their growth, but that endogenous sterol synthesis alone is unable to supply sufficient cholesterol to support maximal rates of steroid hormone secretion. When the cells are subsequently exposed to LDL, the lipoprotein satisfies the sterol requirements for both growth and steroid synthesis; hence, HMG CoA reductase becomes suppressed. That this action requires hydrolysis of the cholesteryl esters of LDL within lysosomes was indicated by the finding that the suppression


608

KOVANEN ET AL.

of HMG CoA reductase was prevented by the lysosomal inhibitor chloroquine. An important question raised by the current studies relates to the mechanism by which the cholesterol of LDL is transported from the lysosome to the mitochondria, where it must undergo side chain cleavage. Recent studies have shown that adrenal cells contain a cytosolic cholesterol-binding protein (31), which might transport cholesterol from lysosomes to mitochondria. Through the use of LDL labeled with [3H]cholesteryl linoleate, it should be possible to demonstrate the occurrence of [3H]cholesterol associated with such a protein in extracts of adrenal cells that have been incubated with [3H]cholesteryl linoleate-labeled LDL. Another important aspect of these studies relates to the specificity of the lipoprotein receptor. As previously demonstrated for mouse adrenal Y-l cells, the receptor in the cultured bovine adrenocortical cells was specific for LDL. HDL did not bind to this receptor, as indicated by its failure to compete effectively with [125I]iodo-LDL for binding or degradation. Moreover, [125I]iodo-HDL was not degraded with high affinity by the bovine adrenocortical cells, a finding that is consistent with similar observations in human fibroblasts (32). As a result of its inability to bind to the LDL receptor or to be degraded with high affinity, HDL was unable to stimulate steroid secretion in the adrenal cells even when added at a cholesterol concentration 10-fold higher than that required for an LDL effect (Table 4). The mechanism by which the rat adrenal uses both HDL and LDL in vivo (5, 6, 11) is not settled by the current studies. It is possible that in vivo some mechanism functions in addition to the LDL receptor to allow adrenocortical cells to utilize the cholesterol of HDL. This mechanism may not be expressed in cell culture. Gwynne et al. have recently reported in preliminary form that primary cultures of rat adrenal cells preferentially utilized the cholesterol of HDL for steroid synthesis, whereas primary cultures of human adrenal cells and cultured Y-l mouse adrenal cells preferentially utilized the cholesterol of LDL (33). It should be noted that the LDL receptor in the bovine adrenocortical cells was subject to metabolic regulation. In particular, the receptor activity was enhanced by 2- to 4-fold when steroid production was stimulated by ACTH or cholera toxin. On the other hand, the receptor was suppressed by high levels of LDL. Thus, the cultured bovine adrenocortical cells, like the human fibroblasts (8, 30), appear to adjust the number of LDL receptors so as to supply only that amount of cholesterol necessary for growth and metabolic purposes. When the bovine adrenocortical cells were cultured in the presence of LDL, the addition of ACTH or cholera toxin caused an increase in LDL receptors (Table 3) without an increase

1979 Endo Vol 104 , No 3

in HMG CoA reductase activity (Table 1) or cholesterol synthesis (Table 6). Thus, when demand for cholesterol is enhanced and LDL is present, the bovine adrenocortical cells preferentially utilize LDL-cholesterol for steroid hormone synthesis while cholesterol synthesis remains suppressed. The functional studies of bovine adrenocortical LDL receptors described in this paper form the basis for studies of LDL receptors in membranes prepared from fresh homogenates of bovine adrenal cortex. These studies are reported in the second paper in this series (17). Acknowledgments We thank Jean Helgeson for her able assistance in the tissue culture laboratory. Gloria Y. Brunschede, Jerry K. Cheek, and Lynda Letzig provided excellent technical assistance. We are grateful to Dr. Celso Gomez-Sanchez for his assistance with the high pressure liquid chromatography.

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LDL RECEPTORS IN BOVINE ADRENAL CELLS 15. Hornsby, P. J., and G. N. Gill, Characterization of adult bovine adrenocortical cells throughout their life span in tissue culture, Endocrinology 102: 926, 1978. 16. Hornsby, P. J., and G. N. Gill, Hormonal control of adrenocortical cell proliferation, J Clin Invest 60: 342, 1977. 17. Kovanen, P. T., S. K. Basu, J. L. Goldstein, and M. S. Brown, Low density lipoprotein receptors in bovine adrenal cortex. II. Low density lipoprotein binding to membranes prepared from fresh tissues, Endocrinology 104: 610, 1979. 18. Brown, M. S., S. E. Dana, and J. L. Goldstein, Regulation of 3hydroxy-3-methylglutaryl coenzyme A reductase activity in cultured human fibroblasts, J Biol Chem 249: 789, 1974. 19. Brown, M. S., and J. L. Goldstein, Suppression of 3-hydroxy-3methylglutaryl coenzyme A reductase activity and inhibition of growth of human fibroblasts by 7-ketocholesterol, J Biol Chem 249: 7306, 1974. 20. Brown, M. S., and J. L. Goldstein, Familial hypercholesterolemia: defective binding of lipoproteins to cultured fibroblasts associated with impaired regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity, Proc Natl Acad Sci 71: 788, 1974. 21. Goldstein, J. L., and M. S. Brown, Binding and degradation of low density lipoproteins by cultured human fibroblasts, J Biol Chem 249: 5153, 1974. 22. Krieger, M., M. S. Brown, J. R. Faust, and J. L. Goldstein, Replacement of endogenous cholesteryl esters of low density lipoprotein with exogenous cholesteryl linoleate, J Biol Chem 253: 4093, 1978. 23. Goldstein, J. L., S. K. Basu, G. Y. Brunschede, and M. S. Brown, Release of low density lipoprotein from its cell surface receptor by sulfated glycosaminoglycans, Cell 7: 85, 1976. 24. Balasubramaniam, S., J. L. Goldstein, J. R. Faust, and M. S. Brown, Evidence for regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and cholesterol synthesis in nonhepatic tissues

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of rat, Proc Natl Acad Sci 73: 2564, 1976. 25. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall, Protein measurement with the Folin phenol reagent, J Biol Chem 193: 265, 1951. 26. Brown, M. S., J. R. Faust, and J. L. Goldstein, Role of the low density lipoprotein receptor in regulating the content of free and esterified cholesterol in human fibroblasts, J Clin Invest 55: 783, 1975. 27. Silber, R. H., Fluorimetric analysis of corticoids, In Glick, D. (ed.), Methods of Biochemical Analysis, vol. 14, Interscience Publishers, New York, 1966, p. 63. 28. Goldstein, J. L., G. Y. Brunschede, and M. S. Brown, Inhibition of the proteolytic degradation of low density lipoprotein in human fibroblasts by chloroquine, concanavalin A, and Triton WR 1339, J Biol Chem 250: 7854, 1975. 29. Goldstein, J. L., S. E. Dana, and M. S. Brown, Esterification of low density lipoprotein cholesterol in human fibroblasts and its absence in homozygous familial hypercholesterolemia, Proc Natl Acad Sci 71: 4288, 1974. 30. Brown, M. S., and J. L. Goldstein, Regulation of the activity of the low density lipoprotein receptor in human fibroblasts, Cell 6: 307, 1975. 31. Lefevre, A., A.-M. Morera, and J. M. Saez, Adrenal cholesterolbinding protein: properties and partial purification, FEBS Lett 89: 287, 1978. 32. Miller, N. E., D. B. Weinstein, and D. Steinberg, Binding, internalization, and degradation of high density lipoprotein by cultured normal human fibroblasts, J Lipid Res 18: 438, 1977. 33. Gwynne, J., B. Hess, R. Rountree, and W. Wolf, Enhancement of steroidogenesis by serum lipoproteins in human, rat, and mouse adrenal cells, Program and Abstracts of the 60th Annual Meeting of The Endocrine Society, June 14-16, Miami, Florida, 1978, p. 221.


Low density lipoprotein receptors in bovine adrenal cortex. II. Low density lipoprotein binding to membranes prepared from fresh tissue.

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Total cholesterol, low density lipoprotein cholesterol, and high density lipoprotein cholesterol and coronary heart disease in Scotland.

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Cholesterol uptake by human glioma cells via receptor-mediated endocytosis of low-density lipoprotein.

A multicentre analysis of four low-density lipoprotein cholesterol direct assays in samples with extreme high-density lipoprotein cholesterol concentrations.

Nonpharmacological approaches for reducing serum low-density lipoprotein cholesterol.

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Binding characteristics of reduced hepatic receptors for acetylated low-density lipoprotein and maleylated bovine serum albumin.

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Reply: Should we forget about low-density lipoprotein cholesterol?

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Should we forget about low-density lipoprotein cholesterol?

The Italian Consensus Conferences on low density lipoprotein-cholesterol apheresis.