Angiotensin II Promotes Selective Uptake of High Density Lipoprotein Cholesterol Esters in Bovine Adrenal Glomerulosa and Human Adrenocortical Carcinoma Cells Through Induction of Scavenger Receptor Class B Type I
NADIA CHERRADI*, MARTINE BIDEAU*, SERGE ARNAUDEAU, NICOLAS DEMAUREX, RICHARD W. JAMES, SALMAN AZHAR, AND ALESSANDRO M. CAPPONI
Division of Endocrinology and Diabetology (N.C., M.B., R.W.J., A.M.C.) and Department of Physiology (Se.A., N.D.), Faculty of Medicine, University Hospital, CH-1211 Geneva, Switzerland; and Geriatric Research, Education, and Clinical Center (Sa.A.), Veterans Affairs Palo Alto Health Care System, Palo Alto, California 94304
Angiotensin II is one of the main physiological regulators of aldosterone biosynthesis in the zona glomerulosa of the ad- renal cortex. The hormone stimulates intracellular choles- terol mobilization to the mitochondrion for steroid biosyn- thesis. Here we have examined whether angiotensin II also modulates exogenous lipoprotein cholesterol ester supply to the steroidogenic machinery and whether this control is ex- erted on the selective transport of high density lipoprotein- derived cholesterol ester to intracellular lipid droplets through the scavenger receptor class B type I. In bovine ad- renal glomerulosa and human NCI H295R adrenocortical car- cinoma cells, high density lipoprotein stimulated steroid pro- duction. Angiotensin II pretreatment for 24 h potentiated this response. Fluorescence microscopy of cellular uptake of re- constituted high density lipoprotein containing a fluorescent cholesterol ester revealed an initial, time-dependent narrow labeling of the cell membrane followed by an intense accu- mulation of the fluorescent cholesterol ester within lipid droplets. At all time points, labeling was more pronounced in cells that had been treated for 24 h with angiotensin II. Flu- orescence incorporation into cells was prevented by a mono-
clonal antibody directed against apolipoprotein A-I. Upon quantitative fluorometric determination, cholesterol ester uptake in angiotensin II-treated bovine cells was increased to 175 ± 15% of controls after 2 h and to 260 + 10% after 4 h of exposure to fluorescent high density lipoprotein. The amount of scavenger receptor class B type I protein detected in cells treated with angiotensin II for 24 h reached 203 + 12% of that measured in control cells (n = 3, P < 0.01). In contrast, low density lipoprotein receptors were only minimally affected by angiotensin II treatment. This increase in scavenger receptor class B type I protein was associated with a 3-fold induction of scavenger receptor class B type I mRNA, which could be prevented by actinomycin D but not by cycloheximide. Sim- ilar results were obtained in the human adenocarcinoma cell line H295R. These observations show that angiotensin II reg- ulates the scavenger receptor class B type I-mediated selec- tive transport of lipoprotein cholesterol ester across the cell membrane as a major source of precursor for mineralocorti- coid biosynthesis in both human and bovine adrenal cells. (Endocrinology 142: 4540-4549, 2001)
P LASMA LOW AND high density lipoproteins are the major source of cholesterol for most steroid hormone- producing tissues (1). The uptake of low density lipoprotein (LDL)-derived cholesterol esters (CE) involves binding of the LDL particles to specific cell surface receptors followed by internalization of the lipoprotein-receptor complex and ly- sosomal hydrolysis leading to the release of CE (2). In con- trast, the delivery of CE from high density lipoproteins (HDL) takes place through a distinct mechanism termed the “selective pathway,” and it involves binding of HDL to the cell surface followed by delivery of CE into the cells without internalization and degradation of the lipoprotein particle (3, 4). The lipid-poor HDL then dissociate from the cells and
reenter the circulation. In adrenocortical cells, lipoprotein- derived CE are stored within lipid droplets. After acute hormonal stimulation, these CE are hydrolyzed to free cholesterol by the cytosolic cholesterol ester hydrolase (5). Cholesterol is then transported to the mitochondrion for its conversion to cortisol and aldosterone.
Until a few years ago, a key missing element in the study of HDL metabolism was a well defined HDL receptor. Recent studies have identified a physiologically relevant membrane receptor for HDL that mediates cholesterol uptake through a selective pathway (6-8). This receptor has been termed scavenger receptor class B type I (SR-BI). SR-BI is believed to bind to the & helical repeats of apolipoprotein A-I (ApoA-I) (7). It has been suggested that this interaction leads to the formation of a nonaqueous channel through which CE move down their concentration gradient to the plasma membrane (7). Because SR-BI has been shown to bind not only HDL but also LDL and modified LDL, it is likely that this receptor may also affect LDL metabolism in vivo (9-12). Tissue distribution studies revealed that SR-BI is expressed in the liver, where
Abbreviations: AngII, Angiotensin II; ApoA-I, apolipoprotein A-I; BODIPY FL C12, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-S-inda- cene-3-dodecanoate; CE, cholesterol ester; h, human; HDL, high density lipoprotein; HDL3, apolipoprotein E-poor high density lipoproteins; LDL, low density lipoprotein; rec-hHDL, cholesteryl BODIPY-high den- sity lipoprotein; SR-BI, scavenger receptor class B type I; SSC, sodium chloride/sodium citrate buffer.
it contributes to the clearance of plasma CE in reverse cho- lesterol transport (4), and in steroidogenic tissues, where it is particularly abundant in the adrenal gland (13). This ex- pression is induced by trophic hormones such as human CG (13), ACTH (14, 15), and LH (13). Also, HDL have been shown to be significantly more effective than LDL in sup- plying cholesterol for corticosterone production in adrenal glands of the rat and mouse (16, 17). HDL has also been reported to be a source of cholesterol for steroidogenesis in human ovarian cells (18). However, the potential role of HDL in mineralocorticoid biosynthesis and its regulation by hor- mones of the cardiovascular system, particularly in the hu- man adrenal gland, have not been addressed adequately.
The aim of the present studies was to examine the ability of bovine adrenal glomerulosa cells and human NCI H295R ad- renocortical cells to selectively take up CE from HDL when challenged with angiotensin II (AngII). In addition, we rea- soned that if SR-BI is a physiologically relevant receptor for HDL-derived CE uptake and provides cholesterol for steroi- dogenesis, its expression might be the target of the same stimuli that enhance cholesterol uptake and aldosterone production. We provide here evidence that this is indeed the case: bovine adrenal glomerulosa cells and human NCI H295R cells effi- ciently internalize HDL-derived CE for aldosterone biosynthe- sis, both in the resting state and under AngII challenge. We also show that SR-BI is expressed in both cell models and that the increase in HDL-derived CE uptake observed upon AngII stim- ulation is accompanied by an increase in SR-BI protein and mRNA levels, demonstrating a coordinated regulation of SR-BI expression and of the intracellular steps leading to adrenal aldosterone biosynthesis.
Materials and Methods
Materials
[Ile5]AngII was purchased from Bachem (Bubendorf, Switzerland). Phosphatidylcholine, sphingomyelin, unesterified cholesterol, and tri- olein were obtained from Sigma (St. Louis, MO). Cholesteryl BODIPY FL C12 (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-S-indacene-3-dode- canoate) was purchased from Molecular Probes, Inc. (Eugene, OR). Anti-SR-BI polyclonal antibody and SR-BI cDNA were generated by one of us (19). Antihuman LDL receptor was supplied by Research Diag- nostic Inc. (Flanders, NJ). Antipregnenolone antiserum was purchased from Biogenesis Ltd. (Poole, UK). Antihuman ApoA-I monoclonal an- tibody was obtained after propagation of the hybridoma cell line H135D3 (ATCC, Manassas, VA). Hybond-N+ membranes, rapid hy- bridization buffer, and the Rediprime random primer labeling kit were supplied by Amersham Pharmacia Biotech (Zurich, Switzerland).
Methods
Bovine adrenal zona glomerulosa cell preparation. Bovine adrenal glands were obtained from a local slaughterhouse. Primary cultures of glo- merulosa cells were prepared by enzymatic dispersion with dispase and purified on Percoll density gradients as described elsewhere (20). Cells were kept in serum-free medium in the absence or the presence of AngII (10 nm) for 24 h before the experiments, which were performed on the 4th day of culture.
Human adrenocortical carcinoma cell culture (NCI H295R). NCI H295R cells were kindly provided by Dr. W.E. Rainey (University of Texas South- western Medical Center, Dallas, TX) and maintained in a 1:1 mixture of DMEM and Ham’s F12 medium containing pyridoxine, L-glutamine, and 15 mM HEPES (Life Technologies, Inc., Basel, Switzerland). The culture medium was supplemented with insulin, transferrin, selenium (ITS+, Becton Dickinson and Co. Labware, Bedford, MA), and Ultroser
(Ligacon, Tagelswangen, Switzerland) as well as with antibiotics as described elsewhere (21).
Human lipoprotein preparation. LDL (density = 1.006-1.063 g/ml) and apolipoprotein E-poor HDL (HDL3; density = 1.125-1.21 g/ml) were isolated from human plasma by sequential ultracentrifugation as de- scribed previously (22). We used exclusively HDL3 to avoid lipoprotein- derived CE uptake via the classic LDL-receptor pathway. Cholesteryl BODIPY-human HDL (rec-hHDL) were reconstituted as described pre- viously (23). In brief, a mixture of egg phosphatidylcholine (2.3 pmol), sphingomyelin (0.6 pmol), fluorescent cholesteryl BODIPY FL C12 as CE (2.24 umol), unesterified cholesterol (0.6 pmol), and triolein (0.34 µmol) was dissolved in chloroform and dried under N2, then resuspended in a 10 mM Tris-HCl buffer (pH 8) containing 150 mM NaCl, 0.25 mM EDTA, and sonicated at 52 C using a Labsonic L sonicator (B. Braun, Melsungen, Germany) equipped with a microtip at a power setting of 30 W. After sonication for 40 min, the temperature was decreased to 42 C and 5 mg of delipidated hHDL3 apolipoprotein in 2 ml of 2.5 M urea was added dropwise. Sonication was continued for an additional 10 min. The son- icated sample was centrifuged for 20 min at 4000 × g and dialyzed against PBS for 6 h at 4 C. Finally, the reconstituted cholesteryl BODIPY- human HDL3 particles were isolated sequentially by ultracentrifugation (16 h, 150,000 × g at 20 C, then 25 h, 150 x g at 20 C, d = 1.21 g/ml) and dialyzed against PBS for 24 h at 4 C. Immediately before use, the prep- aration was dialyzed against serum-free culture medium, and the pro- tein concentration of rec-hHDL3 was determined using the Bio-Rad Laboratories, Inc. AG reagent (Glattbrugg, Switzerland) and BSA as a standard. BODIPY-CE are substrates for acid CE hydrolase (lysosomal) but are not hydrolyzed by the neutral CE hydrolase (cytosolic) (23). Consequently, intracellular fluorescence is considered to be caused by intact CE internalized through a nonendocytic pathway (i.e. a selective pathway).
Fluorescence microscopy. For the study of the uptake and accumulation of rec-hHDL-derived CE, glomerulosa cells were grown on round (25 mm diameter) glass coverslips (0.35 × 106 cells/coverslip) and pretreated with or without AngII (10 nM) for 24 h before incubation with rec-hHDL (30 µg/ml) for periods varying from 5 min to 4 h. After incubation, each coverslip was washed four to five times in Krebs buffer and subse- quently immersed in a thermostatic chamber (Harvard Apparatus, Hol- liston, MA). Cells were immediately imaged on an Axiovert S100TV microscope equipped for epifluorescence microscopy using a 100× (or 40×) oil-immersion objective (numerical aperture, 1.3) (Carl Zeiss, Feld- bach, Switzerland), 488 ± 10 nm excitation (DeltaRam, Photon Tech- nology International, Inc., Monmouth Junction, NJ), a 505DRLP dichroic mirror and a 535RDF40 emission filter (Omega Optical, Brattleboro, VT). Fluorescence emission from the rec-hHDL was captured using a cooled back-illuminated 16-bit charge-coupled device frame transfer camera (Princeton Instruments, Roper Scientific, Trenton, NJ). All of the equip- ment was controlled for image acquisition and analysis with Meta- morph/Metafluor 4.1.2 software (Universal Imaging, West Chester, PA). The fluorescent images were processed with Metamorph in black and white or in pseudocolors.
Fluorescence quantification. Glomerulosa cells were grown on six-well plates (2 × 106 cells/well). After starvation, cells were pretreated with or without AngII (10 nm) for 24 h before incubation with rec-hHDL (30 ug/ml) for periods varying from 5 min to 4 h. After incubation, cells were washed five times in cold PBS containing 0.1% BSA, and then lipids were extracted with hexane-isopropanol (3:2, vol/vol) as described pre- viously (24). Each sample was dried under N2 and then reconstituted with hexane/isopropanol before being transferred to a glass cuvette. Fluorescence was measured at excitation and emission wavelengths 503 and 540 nm, respectively, using a Jasco CAF-110 spectrofluorometer (Jasco Corporation, Tokyo, Japan).
Effect of anti-ApoA-I antibody on rec-hHDL uptake. To test the ability of the anti-ApoA-I antibody to prevent SR-BI-mediated CE uptake, rec-hHDL (30 µg/ml) were preincubated with a monoclonal anti-ApoA-I antibody (120 µg/ml; a generous gift from Dr. Jean-Michel Dayer, Division of Immunology and Allergology, University Hospital, Geneva, Switzer- land) for 30 min at 4 C before being added to bovine glomerulosa cells for 60 min. Fluorescence microscopy and quantification of rec-hHDL uptake were then performed as described above.
Steroid measurement. For steroid production, adrenal glomerulosa cells were grown on 24-well plates (0.5 × 106 cells/well) and preincubated in the absence or the presence of 10 nm AngII for 24 h. Subsequently, cells were exposed or not to AngII and to increasing concentrations of hHDL3 (125 to 500 µg protein/ml) or hLDL (25 to 100 µg protein/ml) for 3 h in serum-free medium. Equivalent masses of CE available to cells in the supplied hHDL3 or hLDL were calculated on the basis of a 5:1 ratio. Pregnenolone was measured by RIA using a commercial antiserum. WIN 19758 (5 µM), an inhibitor of 3ß-hydroxysteroid dehydrogenase, was included in the incubation medium to prevent further metabolism of pregnenolone into progesterone. The aldosterone content of the in- cubation medium was measured by direct RIA using a commercially available kit (DSL Inc., Webster, TX). Pregnenolone and aldosterone production were normalized to milligram cellular proteins.
SDS-PAGE analysis and immunoblotting. SDS-PAGE was performed ac- cording to Laemmli (25). Aliquots from either bovine glomerulosa or NCI H295R cell lysates were resolved by SDS-PAGE and transferred onto a polyvinylidene fluoride membrane (Millipore Corp., Volketswil, Switzerland) that was incubated in blocking buffer (PBS, 0.2% Tween 20, 5% nonfat dry milk) for 2 h and then exposed either to rabbit polyclonal anti-SR-BI antibodies (1:1000 dilution) at 4 C for 2 h or to antihuman LDL receptor antibodies (1:250 dilution) at 4 C overnight. The membrane was thoroughly washed with PBS/Tween buffer (3 × 10 min) and then incubated for 1 h with horseradish peroxidase-labeled goat antirabbit IgG (CovalAb, Oullins, France). The polyvinylidene fluoride sheet was then washed (4 × 15 min), and the antigen-antibody complex was revealed by enhanced chemiluminescence using the Western blotting detection kit from Amersham Pharmacia Biotech and Kodak Biomax film (Rochester, NY).
RNA isolation and Northern blot analysis. Glomerulosa cell total RNA was extracted using the RNAgents kit (Promega Corp., Zurich, Switzerland) according to the instructions of the manufacturer. This system consis- tently yields 50-80 µg of total RNA per 107 cells. For Northern blot analysis, 20-30 µg of RNA was size fractionated on a 1% formaldehyde agarose gel, vacuum transferred onto a Hybond-N+ membrane, and fixed by UV cross-linking. The integrity of the 18s and 28s RNA was checked by ethidium bromide staining of the gel. Hybridization was performed using rat SR-BI cDNA (1.6 kb) generated by RT-PCR of RNA isolated from cAMP-treated rat granulosa cells (19). The cDNA was labeled with [@-32P]dCTP using the Rediprime random primer labeling kit. Northern blots were prehybridized in Rapid Hybridization Buffer at 65 C for 30 min. The @-32P-labeled probe (specific activity, 2 × 106 cpm/ng DNA) was then added, and the incubation was continued for 2 h at 65 C. Blots were washed for 5 and 15 min successively at room temperature in 2X sodium chloride/sodium citrate buffer (SSC), 0.1% SDS, then for 15 min in 1× SSC, 0.1% SDS. The final wash was per- formed at 65 C for 15 min in 1× SSC, 0.1% SDS. RNA-cDNA hybrids were visualized on Kodak Biomax film after a 12- to 24-h exposure period. Blots were stripped and reprobed with mouse glyceraldehyde- 3-phosphate dehydrogenase cDNA (Ambion, Inc. Lugano, Switzerland) to assess RNA loading.
Analysis of data. Results are expressed as means ± SEM. The mean values were compared by ANOVA using Fisher’s test or two-way ANOVA when appropriate. P < 0.05 was considered as statistically significant. Quantitation of autoradiograms was performed using a Molecular Dy- namics, Inc. (Sunnyvale, CA) Computing Densitometer and Image- Quant software.
Results
Steroidogenic response of bovine glomerulosa cells and NCI H295R cells to lipoproteins
To examine whether bovine adrenal glomerulosa cells can use HDL-derived CE for steroid production, we incubated cells that had been pretreated with or without 10 nm AngII for 24 h with increasing concentrations of hHDL3 for 3 h, and the production of pregnenolone and aldosterone was mea- sured in the incubation medium. As shown in Fig. 1A, non- stimulated cells responded to hHDL3 with a concentration-
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dependent increase in pregnenolone production, which reached approximately 15-fold the basal values. Similarly, aldosterone production was stimulated 16-fold at 500 µg/ml hHDL3. This suggested that HDL can deliver substantial amounts of cholesterol esters to glomerulosa cells to support mineralocorticoid synthesis. Furthermore, AngII-pretreated cells showed a concentration-dependent increase in preg- nenolone and aldosterone formation that was approximately 3- and 6-fold greater than that observed in nonpretreated cells, respectively (Fig. 1A). We then compared the steroi- dogenic response of glomerulosa cells supplied with hHDL3 to that of cells exposed to hLDL. As shown in Fig. 1B, no significant differences were observed in the steroidogenic response to both lipoprotein species, either in the basal state or after AngII pretreatment, a result suggesting that hHDL3 and hLDL were equally effective.
We next examined the effect of hHDL3 (250 µg/ml) on aldosterone biosynthesis in control and AngII-stimulated hu- man NCI H295R cells during a 24-h period of incubation (Fig. 1C). hHDL3 enhanced aldosterone production by 1.6-fold in both control and AngII-stimulated cells (n = 6).
Uptake and intracellular accumulation of fluorescent CE in bovine glomerulosa cells and human NCI H295R cells
We next examined the uptake and storage of HDL3- derived CE within these cells, using hHDL3 reconstituted with the nonhydrolyzable fluorescent CE, rec-hHDL3. Figure 2 shows fluorescence images of time-dependent CE uptake in intact bovine adrenal glomerulosa cells that had been pretreated in the absence (panels b-d) or in the presence (panels f-h) of AngII for 24 h. In the basal state and in the absence of rec-HDL3 (Fig. 2, a and e), glomerulosa cells dis- play a faint fluorescence, presumably attributable to intrinsic lipid droplets. In control cells, a narrow labeling of the plasma membrane appeared within 30-60 min of exposure to rec-HDL3. By 1-2 h of exposure to rec-HDL3, control cells had accumulated massive amounts of fluorescent CE within lipid droplets and increased membrane, cytosolic, and pe- rinuclear signals were observed. Importantly, at all time points, the labeling in AngII-pretreated cells was more in- tense compared with that in the corresponding control cells (panels f-h vs. b-d). In Fig. 2i, AngII-prestimulated glo- merulosa cells are shown at a higher magnification after 4 h of exposure to rec-HDL3. The numerous highly fluorescent spots correspond in size, location, and number to lipid drop- lets, as confirmed by a Nomarski image of the same cells (Fig. 2j).
Images from similar experiments conducted in human NCI H295R cells are presented in Fig. 2, panels k to r. These cells internalized substantial amounts of hHDL3-derived CE in the basal state in a time-dependent manner (Fig. 2, 1-n), and this process was markedly enhanced after a 24-h pre- treatment with AngII (Fig. 2, p-r).
The major apolipoprotein component of HDL3 is ApoA-I. Recent studies in ApoA-I knockout mice suggested that ApoA-I may play a crucial role in the delivery of HDL cho- lesterol to steroidogenic tissues (26). We preincubated rec- HDL3 with a monoclonal antibody against ApoA-I before adding it to glomerulosa cells for 60 min. Fluorescence mi-
croscopy analysis revealed that HDL3-derived CE uptake was markedly reduced in control cells in the presence of the anti-ApoA-I antibody (Fig. 2, s and t). Similar results were obtained in AngII-pretreated cells (Fig. 2, u and v), suggest- ing that a crucial event in HDL3-derived CE uptake is me- diated by ApoA-I.
To further substantiate these qualitative results, we next quantified the amounts of CE taken up by glomerulosa cells using fluorometry. As shown in Fig. 3, a time-dependent increase of CE uptake was observed in control cells. In agree- ment with the fluorescence imaging data, AngII pretreat- ment significantly increased CE uptake, reaching 175 ± 15% of controls after 2 h of exposure to rec-HDL (n = 3, P < 0.01) and 271 ± 8% after 4 h (n = 3; data not shown). Qualitatively similar results were obtained in human NCI H295R cells: AngII pretreatment enhanced CE uptake to 165 ± 8% of controls (n = 3, P < 0.01) after a 4-h exposure to rec-hHDL3. When rec-hHDL3 were first incubated with the anti-ApoA-I antibody, fluorometric measurements revealed that CE up- take was reduced by 41 ± 3% (n = 3, P < 0.05 vs. controls with no antiserum) in both control and AngII-pretreated bovine glomerulosa cells. In contrast, when we used a non- relevant monoclonal antibody directed against apolipopro- tein E instead of the anti-ApoA-I antibody, AngII-induced BODIPY-CE uptake was not significantly affected (86 ± 6% of controls with no antibody, n = 3, NS).
Effect of AngII on SR-BI protein expression
We next examined whether the expression of the HDL receptor SR-BI was modulated by AngII. The Western blots shown in Fig. 4A demonstrate the kinetics of expression of SR-BI protein in bovine glomerulosa cells incubated in the absence (basal) or in the presence of AngII. An immunore- active band of approximately 86 kDa, a molecular mass sim- ilar to that described for murine SR-BI (6), was detected. In control cells, steady state levels of SR-BI expression were maintained for at least 24 h. AngII treatment caused a time- dependent increase in SR-BI, reaching 203 ± 12% of control values (n = 4) after 24 h of stimulation (Fig. 4B). This effect of AngII was concentration dependent between 10-10 and 10-8 M (Fig. 4C).
To examine the specificity of the effect of AngII on SR-BI expression, we compared SR-BI and LDL receptor expression in human NCI H295R cells after AngII treatment. Western blot analysis revealed that both receptors were coexpressed in this cell line (Fig. 5). AngII increased SR-BI protein ex- pression by 230 ± 15% of control values after 24 h (Fig. 5A; n = 3, P < 0.001), whereas LDL receptor expression was only modestly affected by AngII (122 + 3% of control values; n = 3, NS; Fig. 5B). In contrast, stimulation of NCI H295R cells with dibutyryl cAMP for 24 h resulted in a more pronounced induction of LDL receptor expression (180 ± 4% of control values; n = 3, P < 0.001; Fig. 5B).
Effect of AngII on SR-BI mRNA steady state levels
To determine whether the AngII-induced increase in SR-BI protein expression reflects changes in SR-BI mRNA levels, total RNA was analyzed by Northern blotting. Fig. 6A shows that a transcript of 2.8 kb was detected. Densitometric anal-
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ysis indicates that AngII induced a 3-fold increase in SR-BI mRNA levels compared with control values (n = 4; Fig. 6B). Cycloheximide did not affect the AngII-elicited increase in SR-BI mRNA, suggesting that transactivating factors are al- ready present within the cell. However, SR-BI mRNA in- duction by AngII was abolished by actinomycin D, suggest- ing a transcriptional control of SR-BI expression by AngII. When glomerulosa cells were incubated for 24 h in the pres- ence of hHDL3, a significant increase (152 ± 4%; P < 0.05) in SR-BI mRNA was observed. Finally, after a combined treat- ment with both AngII and hHDL3, the transcriptional effect of AngII on SR-BI mRNA levels was maintained. Very sim- ilar results were obtained in human NCI H295R cells. SR-BI mRNA levels were increased to 188 ± 13% over controls after a 24-h exposure to AngII (n = 3, P < 0.01). This increase was not significantly altered by cycloheximide but was com- pletely prevented by actinomycin D. The addition of HDL3 did not affect AngII-elicited induction of SR-BI mRNA in human NCI H295R cells.
Discussion
LDL are the major cholesterol-carrying circulating li- poproteins in humans, and the LDL receptor-mediated path- way is highly developed in this species. As a consequence, the role of HDL as a potential source of cholesterol to human cells has received less attention for several years and has remained elusive. The recent discovery that SR-BI mediates both HDL binding and HDL-derived CE selective uptake has provided an important link between the selective uptake pathway and a specific cell surface receptor (6, 27). These initial observations have led to extensive studies on the reg- ulation of both SR-BI expression and HDL-derived CE selective uptake by trophic hormones and activators of adenylyl cyclase in steroidogenic tissues, particularly in the
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adrenal gland and the gonads (13, 18, 19, 28-35). In the present study, we have investigated whether HDL could also supply cholesterol to the human adrenocortical carcinoma cell line NCI H295R and to bovine adrenal glomerulosa cells challenged with the major physiological regulator of aldo- sterone biosynthesis, the Ca2+-mobilizing hormone AngII.
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Three major conclusions can be drawn from the present studies. 1) Human NCI H295R cells and bovine adrenal glo- merulosa cells do indeed “selectively” internalize and pro- cess HDL-derived CE as a significant source of precursor for steroid hormone biosynthesis. 2) AngII enhances HDL- derived CE uptake as well as SR-BI expression (protein and mRNA) in both cell types, confirming a tight link between the selective uptake, the HDL receptor SR-BI, and steroidogen- esis. 3) In contrast to cAMP, which has been shown to exert
a more pronounced induction on LDL receptors than on SR-BI protein in human adrenal cells (34), AngII behaves in the opposite manner, potently increasing SR-BI expression while barely affecting LDL receptors. This finding suggests the possibility that the cells may switch between HDL and LDL utilization depending on the type of agonist that chal- lenges them.
The demonstration of efficient HDL-derived CE uptake supporting steroidogenesis in glomerulosa cells is based on functional and fluorescence data. Using HDL as CE donors, we show that a substantial increase in pregnenolone and aldosterone production occurs in bovine adrenal glomeru- losa cells and human NCI H295R cells. Of importance here is the finding that utilization of HDL3-derived CE occurs even in the resting state. This result is in contrast to obser- vations made in human and rat ovarian granulosa cells, in which selective CE uptake is dependent on gonadotropin stimulation (18, 23, 36). Furthermore, AngII and lipoproteins act synergistically in supporting steroidogenesis, as indi- cated by the fact that the combined addition of AngII and HDL3 is clearly more effective than either agent alone. We have previously demonstrated that AngII increases the mo- bilization of cholesterol to the inner mitochondrial mem- brane (37). In bovine adrenal fasciculata cells, ACTH-stim- ulated cortisol production was significantly greater in the presence of HDL compared with LDL (29). We report here that HDL and LDL are equally effective in supporting ste- roidogenesis in bovine glomerulosa cells, indicating that both the endocytic and selective pathways are functional in these cells. However, the contribution of SR-BI to LDL- derived CE selective uptake in bovine and human adrenal cells remains to be determined. Indeed, SR-BI has been shown to mediate the efficient uptake of LDL-derived CE via a selective uptake mechanism in Y1 adrenocortical cells and in SR-BI-transfected COS-7 cells (12) as well as in Chinese hamster ovary cells (10, 11). Furthermore, ovarian granulosa cells from LDL receptor knockout mice selectively internalize LDL, suggesting that SR-BI may also affect the metabolism of LDL in vivo (38). Most importantly, Leitersdorf et al. (39) have reported in earlier studies that AngII induces prefer- ential uptake of the CE moiety over the protein moiety of LDL in bovine adrenal glomerulosa cells.
The BODIPY-CE fluorescence microscopy experiments al- lowed us to directly follow the fate of labeled CE within the cells. HDL3-derived CE uptake is a rapid process, visible within 5 min, in both bovine glomerulosa cells and human NCI H295R cells. In contrast to ovarian granulosa cells, in which the plasma membrane labeling has been reported to be minimal (40), the labeling of the cell membrane in both adrenal glomerulosa and human NCI H295R cells is pro- nounced and persistent, a finding consistent with the obser- vation that the receptor concentrates in plasma membrane microdomains called caveolae (41, 42). Interestingly, most of the fluorescent CE accumulates within lipid droplets in areas essentially situated under the plasma membrane, where large numbers of mitochondria can be found (43, 44).
The fluorometric experiments allowed us to precisely quantitate total CE uptake while overcoming intrinsic diffi- culties of the microscopic approach, such as photobleaching of the probe (40). AngII pretreatment doubled HDL-derived
CE uptake, both in glomerulosa cells and in human NCI H295R cells. These results are of the same order of magnitude as those obtained with [3H]CE-labeled HDL in human NCI H295R cells prestimulated with 8-bromo-cAMP (34) and in Leydig cells treated with human CG (35). In contrast, the selective uptake was found to be dramatically increased in rat ovarian granulosa cells stimulated with dibutyryl cAMP, reaching 10- to 15-fold the value measured in nonstimulated cells (23).
The specificity of the SR-BI-mediated CE uptake in adrenal cells was further demonstrated with an anti-ApoA-I anti- body. Recent studies of CE selective uptake in steroidogenic tissues of ApoA-I knockout mice stress that ApoA-I plays a critical role in this process (26, 45). Using an antibody against ApoA-I, we demonstrate a significant reduction (41%) of HDL3-derived CE uptake in bovine glomerulosa cells stim- ulated or not with AngII. This finding is consistent with data showing that 40% of the ApoA-I bound to SR-BI can be chemically cross-linked to the receptor (7).
Our study demonstrates a tight association between the function of the selective pathway and SR-BI expression, both processes being increased to the same extent by AngII. SR-BI has been shown to be up-regulated by ACTH in the murine adrenal gland in vivo and in Y1BS1 adrenal cells in vitro, as well as by dibutyryl cAMP in ovarian granulosa cells and by 8-bromo-cAMP in human adrenal cells (14, 19, 32-34, 46). We report here, for the first time, that SR-BI is induced to a comparable level in bovine and human adrenocortical cells after challenge with AngII in a concentration-dependent manner. Importantly, although LDL-derived CE may be taken up by the cell through both the endocytic and selective pathways, the major impact of AngII stimulation is to in- crease preferentially the expression of SR-BI and not that of the LDL receptor and thereby to promote the selective uptake of CE. Our data indicate that LDL receptor expression is more sensitive to the cAMP signaling pathway than to the Ca2+- messenger system. This finding is in line with previous data showing that the level of induction of SR-BI expression by cAMP was consistently lower than that of the LDL receptor in human NCI H295R cells (34).
The increase in SR-BI protein content after a 24-h exposure to AngII was accompanied by a similar induction of SR-BI mRNA levels. This concomitant increase in SR-BI protein and mRNA has been reported in various steroidogenic cell types from different species after stimulation with activators of the cAMP pathway, suggesting a conservation between species of SR-BI regulation (13, 15, 28, 30, 34). The induction of SR-BI mRNA by AngII does not require de novo protein synthesis, similar to what has been observed in NCI H295R cells ex- posed to ACTH (34), indicating that the effects of AngII are independent of short-lived proteins. Moreover, actinomycin D abolished the increase in SR-BI mRNA triggered by AngII, suggesting a transcriptional control of the SR-BI gene by the hormone, as has been reported for ACTH in human adrenal cells (34). The transcription factor steroidogenic factor 1 (SF-1) has been reported to mediate the transcription of hu- man and murine SR-BI genes by cAMP (47, 48). The potential role of SF-1 in AngII-induced transcription of the SR-BI gene will require further analysis.
An important question is whether the AngII-induced in-
crease in SR-BI expression in both bovine glomerulosa cells and human NCI H295R cells is secondary to hormone- mediated changes in cellular cholesterol homeostasis or is the result of direct effects of AngII on SR-BI gene expression. Two sets of data support the latter hypothesis. First, in sep- arate experiments, we have observed that AngII still in- creased SR-BI mRNA levels when glomerulosa cells were treated with aminogluthetimide to prevent cholesterol side chain cleavage and thereby cholesterol depletion (data not shown). This result speaks in favor of a direct effect of AngII on the SR-BI gene independent of cholesterol status. Con- sequently, an involvement of transcription factors such as sterol regulatory element binding proteins in AngII stimu- lation of SR-BI expression in bovine and human adrenocor- tical cells may be ruled out, although sterol regulatory ele- ments have been described in the SR-BI promoter (49). It is worth mentioning that a sterol-independent regulatory ele- ment that binds the C/EBP transcription factor has been identified recently in the human LDL receptor promoter (50), in spite of the well documented regulation of this receptor by cellular cholesterol status and sterol regulatory element binding proteins (51, 52). Whether such a response element is also present in the SR-BI promoter is not known. Second, we found that the up-regulation of SR-BI by AngII is insen- sitive to HDL3 loading. This result is similar to that obtained in luteinized granulosa cells incubated with HDL (19) but is in contrast to what has been observed in other cell systems, in which LDL induce a down-regulation of the LDL receptor and other cholesterol-sensitive genes (53-55).
In conclusion, the present study shows that bovine and, more importantly, human adrenocortical cells take up mas- sive amounts of HDL-derived CE as a substrate for steroid hormone production in both the basal state and under AngII stimulation. Although blood lipoprotein profiles in human and cow are in favor of LDL (56), and although several earlier reports have concluded that HDL are not effective choles- terol donors for human and bovine steroidogenic cells (17,57, 58), our data clearly indicate that adrenocortical cells from these species efficiently take up and metabolize HDL- derived CE to support AngII-induced aldosterone biosyn- thesis. This finding is in keeping with in vivo studies of adrenal function in familial hypercholesterolemia and hy- pobeta-lipoproteinemia, which have suggested a potential role for HDL in human adrenocortical cholesterol meta- bolism (59, 60).
Acknowledgments
The authors are grateful to Andres Maturana for helpful discussions. We thank Manuella Rey, Silvana Bioletto, Barbara Kalix, and Marie- Claude Brulhart for their excellent technical assistance.
Received February 8, 2001. Accepted June 11, 2001.
Address all correspondence and requests for reprints to: Prof. Ales- sandro M. Capponi, Division of Endocrinology and Diabetology, Uni- versity Hospital, 24 rue Micheli-du-Crest, CH-1211 Geneva 14, Swit- zerland. E-mail: capponi@cmu.unige.ch.
This work was supported by Swiss National Science Foundation Grant 31.52779-97 (to A.M.C.) and by the Office of Research and De- velopment, Medical Research Service, Department of Veterans Affairs (to Sa.A.).
* These two authors contributed equally to this work.
References
1. Gwynne JT, Strauss JF 1982 The role of lipoproteins in steroidogenesis and cholesterol metabolism in steroidogenic glands. Endocr Rev 3:299-329
2. Brown MS, Goldstein JL 1986 A receptor-mediated pathway for cholesterol homeostasis. Science 232:34-47
3. Glass C, Pittman RC, Weinstein DB, Steinberg D 1983 Dissociation of tissue uptake of cholesterol ester from that of apoprotein A-I of rat plasma high density lipoprotein: selective delivery of cholesterol ester to liver, adrenal, and gonad. Proc Natl Acad Sci USA 80:5435-5439
4. Pieters MN, Schouten D, Van Berkel TJ 1994 In vitro and in vivo evidence for the role of HDL in reverse cholesterol transport. Biochim Biophys Acta 1225:125-134
5. Mikami K, Nishikawa T, Saito Y, et al. 1984 Regulation of cholesterol me- tabolism in rat adrenal glands: effect of adrenocorticotropin, cholesterol, and corticosteroids on acyl-coenzyme A synthetase and cholesterol ester hydrolase. Endocrinology 114:136-140
6. Acton S, Rigotti A, Landschulz KT, Xu S, Hobbs HH, Krieger M 1996 Identification of scavenger receptor SR-B1 as a high density lipoprotein re- ceptor. Science 271:520
7. Williams DL, Connelly MA, Temel RE, et al. 1999 Scavenger receptor BI and cholesterol trafficking. Curr Opin Lipidol 10:329-339
8. Rigotti A, Trigatti B, Babitt J, Penman M, Xu S, Krieger M 1997 Scavenger receptor BI: a cell surface receptor for high density lipoprotein. Curr Opin Lipidol 8:181-188
9. Acton SL, Scherer PE, Lodish HF, Krieger M 1994 Expression cloning of SR-BI, a CD36-related class B scavenger receptor. J Biol Chem 269:21003-21009
10. Stangl H, Cao G, Wyne KL, Hobbs HH 1998 Scavenger receptor, class B, type I-dependent stimulation of cholesterol esterification by high density lipopro- teins, low density lipoproteins, and nonlipoprotein cholesterol. J Biol Chem 273:31002-31008
11. Stangl H, Hyatt M, Hobbs HH 1999 Transport of lipids from high and low density lipoproteins via scavenger receptor-BI. J Biol Chem 274:32692-32698
12. Swarnakar S, Temel RE, Connelly MA, Azhar S, Williams DL 1999 Scavenger receptor class B, type I, mediates selective uptake of low density lipoprotein cholesteryl ester. J Biol Chem 274:29733-29739
13. Landschulz KT, Pathak RK, Rigotti A, Krieger M, Hobbs HH 1996 Regulation of scavenger receptor, class B, type I, a high density lipoprotein receptor, in liver and steroidogenic tissues of the rat. J Clin Invest 98:984-995
14. Rigotti A, Edelmann ER, Seifert P, et al. 1996 Regulation by adrenocortico- tropic hormone of the in vivo expression of scavenger receptor class B type I (SR-BI), a high density lipoprotein receptor, in steroidogenic cells of the murine adrenal gland. J Biol Chem 271:33545-33549
15. Liu J, Voutilainen R, Heikkilä P, Kahri AI 1997 Ribonucleic acid expression of the CLA-1 gene, a human homolog to mouse high density lipoprotein receptor SR-BI, in human adrenal tumors and cultured adrenal cells. J Clin Endocrinol Metab 82:2522-2527
16. Gwynne JT, Mahaffee D, Brewer HB, Ney RL 1976 Adrenal cholesterol uptake from plasma lipoproteins: regulation by corticotropin. Proc Natl Acad Sci USA 73:4329-4333
17. Kovanen PT, Schneider WJ, Hillman GM, Goldstein JL, Brown MS 1979 Separate mechanisms for the uptake of high and low density lipoproteins by mouse adrenal gland in vivo. J Biol Chem 254:5498-5505
18. Azhar S, Tsai L, Medicherla S, Chandrasekher Y, Giudice L, Reaven E 1998 Human granulosa cells use high density lipoprotein cholesterol for steroido- genesis. J Clin Endocrinol Metab 83:983-991
19. Azhar S, Nomoto A, Leers-Sucheta S, Reaven E 1998 Simultaneous induction of an HDL receptor protein (SR-BI) and the selective uptake of HDL-cholesteryl esters in a physiologically relevant steroidogenic cell model. J Lipid Res 39: 1616-1628
20. Python CP, Laban OP, Rossier MF, Vallotton MB, Capponi AM 1995 The site of action of Ca2+ in the activation of steroidogenesis: studies in Ca2+-clamped bovine adrenal zona-glomerulosa cells. Biochem J 305:569-576
21. Bird IM, Hanley NA, Word RA, et al. 1993 Human NCI-H295 adrenocortical carcinoma cells: a model for angiotensin-II-responsive aldosterone secretion. Endocrinology 133:1555-1561
22. James RW, Proudfoot A, Pometta D 1989 Immunoaffinity fractionation of high-density lipoprotein subclasses 2 and 3 using anti-apolipoprotein A-I and A-II immunosorbent gels. Biochim Biophys Acta 1002:292-301
23. Reaven E, Tsai L, Azhar S 1995 Cholesterol uptake by the “selective” pathway of ovarian granulosa cells: early intracellular events. J Lipid Res 36:1602-1617
24. Radin NS 1981 Extraction of tissue lipids with a solvent of low toxicity. Methods Enzymol 72:5-7
25. Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 27:680-685
26. Plump AS, Erickson SK, Weng W, Partin JS, Breslow JL, Williams DL 1996 Apolipoprotein A-I is required for cholesteryl ester accumulation in steroi- dogenic cells and for normal adrenal steroid production. J Clin Invest 97: 2660-2671
27. Temel RE, Trigatti B, Demattos RB, Azhar S, Krieger M, Williams DL 1997 Scavenger receptor class B, type I (SR-BI) is the major route for the delivery of
high density lipoprotein cholesterol to the steroidogenic pathway in cultured mouse adrenocortical cells. Proc Natl Acad Sci USA 94:13600-13605
28. Rajapaksha WR, McBride M, Robertson L, O’Shaughnessy PJ 1997 Sequence of the bovine HDL-receptor (SR-BI) cDNA and changes in receptor mRNA expression during granulosa cell luteinization in vivo and in vitro. Mol Cell Endocrinol 134:59-67
29. Yaguchi H, Tsutsumi K, Shimono K, Omura M, Sasano H, Nishikawa T 1998 Involvement of high density lipoprotein as substrate cholesterol for steroido- genesis by bovine adrenal fasciculo-reticularis cells. Life Sci 62:1387-1395
30. Li X, Peegel H, Menon KMJ 1998 In situ hybridization of high density li- poprotein (scavenger, type I) receptor messenger ribonucleic acid (mRNA) during folliculogenesis and luteinization: evidence for mRNA expression and induction by human chorionic gonadotropin specifically in cell types that use cholesterol for steroidogenesis. Endocrinology 139:3043-3049
31. Reaven E, Nomoto A, Leers-Sucheta S, Temel RE, Williams DL, Azhar S 1998 Expression and microvillar localization of scavenger receptor class B, type I (a high density lipoprotein receptor) in luteinized and hormone-desensitized rat ovarian models. Endocrinology 139:2847-2856
32. Reaven E, Lua Y, Nomoto A, et al. 1998 The selective pathway and a high- density lipoprotein receptor (SR-BI) in ovarian granulosa cells of the mouse. Biochim Biophys Acta 1436:565-576
33. Sun Y, Wang N, Tall AR 1999 Regulation of adrenal scavenger receptor-BI expression by ACTH and cellular cholesterol pools. J Lipid Res 40:1799-1805
34. Martin G, Pilon A, Albert C, et al. 1999 Comparison of expression and regulation of the high-density lipoprotein receptor SR-BI and the low-density lipoprotein receptor in human adrenocortical carcinoma NCI-H295 cells. Eur J Biochem 261:481-491
35. Reaven E, Zhan L, Nomoto A, Leers-Sucheta S, Azhar S 2000 Expression and microvillar localization of scavenger receptor class B, type I (SR-BI) and se- lective cholesteryl ester uptake in Leydig cells from rat testis. J Lipid Res 41:343-356
36. Azhar S, Tsai L, Reaven E 1990 Uptake and utilization of lipoprotein cho- lesteryl esters by rat granulosa cells. Biochim Biophys Acta 1047:148-160
37. Cherradi N, Rossier MF, Vallotton MB, et al. 1997 Submitochondrial distri- bution of three key steroidogenic proteins (steroidogenic acute regulatory protein, P450 side-chain cleavage and 3-hydroxysteroid dehydrogenase isomerase enzymes) upon stimulation by intracellular calcium in adrenal glo- merulosa cells. J Biol Chem 272:7899-7907
38. Azhar S, Luo Y, Medicherla S, Reaven E 1999 Upregulation of selective cholesteryl ester uptake pathway in mice with deletion of low-density lipopro- tein receptor function. J Cell Physiol 180:190-202
39. Leitersdorf E, Stein O, Stein Y 1985 Angiotensin II stimulates receptor- mediated uptake of LDL by bovine adrenal cortical cells in primary culture. Biochim Biophys Acta 835:183-190
40. Reaven E, Tsai L, Azhar S 1996 Intracellular events in the “selective” transport of lipoprotein-derived cholesteryl esters. J Biol Chem 271:16208-16217
41. Babitt J, Trigatti B, Rigotti A, et al. 1997 Murine SR-BI, a high density li- poprotein receptor that mediates selective lipid uptake, is N-glycosylated and fatty acylated and colocalizes with plasma membrane caveolae. J Biol Chem 272:13242-13249
42. Graf GA, Connell PM, van der Westhuyzen DR, Smart EJ 1999 The class B, type I scavenger receptor promotes the selective uptake of high density li- poprotein cholesterol ethers into caveolae. J Biol Chem 274:12043-12048
43. Rossier MF 1997 Confinement of intracellular calcium signaling in secretory and steroidogenic cells. Eur J Endocrinol 137:317-325
44. Brandenburger Y, Arrighi JF, Rossier MF, Maturana A, Vallotton MB, Cap-
poni AM 1999 Measurement of perimitochondrial Ca2+ concentration in bo- vine adrenal glomerulosa cells with aequorin targeted to the outer mitochon- drial membrane. Biochem J 341:745-753
45. Xu S, Laccotripe M, Huang X, Rigotti A, Zannis VI, Krieger M 1997 Apoli- poproteins of HDL can directly mediate binding to the scavenger receptor SR-BI, an HDL receptor that mediates selective lipid uptake. J Lipid Res 38:1289-1298
46. Wang N, Weng W, Breslow JL, Tall AR 1996 Scavenger receptor BI (SR-BI) is up-regulated in adrenal gland in apolipoprotein A-I and hepatic lipase knock- out mice as a response to depletion of cholesterol stores: in vivo evidence that SR-BI is a functional high density lipoprotein receptor under feedback control. J Biol Chem 271:21001-21004
47. Cao G, Garcia CK, Wyne KL, Schultz RA, Parker KL, Hobbs HH 1997 Structure and localization of the human gene encoding SR-BI/CLA-1: evidence for transcriptional control by steroidogenic factor 1. J Biol Chem 272:33068- 33076
48. Lopez D, Sandhoff TW, McLean MP 1999 Steroidogenic factor-1 mediates cyclic 3’,5’-adenosine monophosphate regulation of the high density lipopro- tein receptor. Endocrinology 140:3034-3044
49. Lopez D, McLean MP 1999 Sterol regulatory element-binding protein-1a binds to cis elements in the promoter of the rat high density lipoprotein receptor SR-BI gene. Endocrinology 140:5669-5681
50. Liu J, Ahlborn TE, Briggs MR, Kraemer FB 2000 Identification of a novel sterol-independent regulatory element in the human low density lipoprotein receptor promoter. J Biol Chem 275:5214-5221
51. Wang X, Briggs MR, Hua X, Yokoyama C, Goldstein JL, Brown MS 1993 Nuclear protein that binds sterol regulatory element of low density lipoprotein receptor promoter. II. Purification and characterization. J Biol Chem 268:14497- 14504
52. Wang X, Sato R, Brown MS, Hua X, Goldstein JL 1994 SREBP-1, a membrane- bound transcription factor released by sterol-regulated proteolysis. Cell 77: 53-62
53. Medicherla S, Azhar S, Cook DA, Reaven E 1996 Regulation of cholesterol responsive genes in ovary cells: impact of cholesterol delivery systems. Bio- chemistry 35:6243-6250
54. Reaven E, Tsai L, Spicher M, et al. 1994 Enhanced expression of granulosa cell low density lipoprotein receptor activity in response to in vitro culture con- ditions. J Cell Physiol 161:449-462
55. Russell DW, Yamamoto T, Schneider WJ, Slaughter CJ, Brown MS, Gold- stein JL 1983 cDNA cloning of the bovine low density lipoprotein receptor: feedback regulation of a receptor mRNA. Proc Natl Acad Sci USA 80:7501-7505
56. Gwynne JT, Mahaffee DD 1986 Lipoproteins and steroid hormone-producing tissues. Methods Enzymol 129:679-690
57. Higashijima M, Nawata H, Kato H, Ibayashi H 1987 Studies on lipoprotein and adrenal steroidogenesis. I. Roles of low density lipoprotein- and high density lipoprotein-cholesterol in steroid production in cultured human ad- renocortical cells. Endocrinol Jpn 34:635-645
58. Kovanen PT, Faust JR, Brown MS, Goldstein JL 1979 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. Endocrinology 104:599-609
59. Laue L, Hoeg JM, Barnes K, Loriaux DL, Chrousos GP 1987 The effect of mevinolin on steroidogenesis in patients with defects in the low density li- poprotein receptor pathway. J Clin Endocrinol Metab 67:531-535
60. Ilingworth DR, Kenny TA, Orwoll ES 1982 Adrenal function in heterozygous and homozygous hypobetalipoproteinemia. J Clin Endocrinol Metab 54:27-33