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Adrenocortical Lipid Depletion Gene (ald) in AKR Mice Is Associated with an Acyl-CoA:Cholesterol Acyltransferase (ACAT) Mutation*

ald, a recessive allele in AKR inbred mice, is responsible for complete adrenocortical lipid depletion in postpubertal males, which appears to be androgen dependent. Two recent observations (adrenocortical lipid depletion in acyl-CoA:cholesterol acyltransferase-deficient (Acact−/−) mice and the mapping of Acact to a region of chromosome 1 containing theald locus) prompted us to ask whether adrenocortical lipid depletion in AKR mice results from an Acact mutation. Refined genetic mapping of Acact and ald was consistent with colocalization of these loci. CrossingAcact−/− with AKR (ald/ald) mice yielded postpubertal male offspring characterized by adrenocortical lipid depletion, indicating that these loci are not complementational and are therefore allelic. Immunoblotting of preputial gland homogenates demonstrated that AKR mice had an ACAT protein with a lower molecular mass than other mouse strains. Analysis of Acact cDNA from AKR mice revealed a deletion of the first coding exon and two missense mutations. Despite these coding sequence differences, the ACAT protein from the ald allele catalyzed cholesterol esterification activity at levels similar to that of wild-type protein. We speculate that the adrenocortical lipid depletion resulting from theald mutation is caused by an altered susceptibility of the mutant protein to modifying factors, such as androgen production at puberty, in an as yet undetermined manner.

About a dozen naturally occurring mutations that affect lipid metabolism in mice have been described (1). In most cases, the mutant genes have not yet been cloned. One such mutation, adrenocortical lipid depletion (ald), 1 is expressed as a recessive trait in inbred AKR mice and was first described in 1955 by Arnesen (2,3). The ald phenotype is characterized by spontaneous lipid depletion in the zona fasciculata at the time of sexual maturation (2,4). The lipid depletion results from a loss of cholesterol esters, which are normally abundant in the adrenocortical cells, but not of free cholesterol or triacylglycerols. In male ald/ald mice, the lipid depletion is nearly total and appears to be androgen-dependent (5). Male ald/ald mice younger than 30 days have normal adrenocortical lipid content, but cholesterol ester stores are rapidly lost between 30 and 40 days, and the depletion is essentially complete by 50 days (5). In female mice, the lipid depletion is subtotal and variable (4). Prepubertal orchiectomy prevents the ald phenotype in male AKR mice, and the phenotype has been reversed by postpubertal orchiectomy (6) or by the administration of adrenocorticotropic hormone (2) or cortisone (7). The ald locus was mapped to chromosome 1 in 1976 (8). The DBA/2 strain is characterized by a similar adrenocortical lipid depletion phenotype, which appears to be due to a different genetic locus (6).
We have recently generated mice in which we have disrupted the gene encoding acyl-CoA:cholesterol acyltransferase (ACAT) (9), an enzyme responsible for catalyzing the formation of cholesterol esters from free cholesterol and fatty acids (10 -12). ACAT-deficient (AcactϪ/Ϫ) mice have tissue-specific reductions in cholesterol esters, including near-total depletion of cholesterol esters in the adrenal cortex in both males and females (9). Coincident with these observations, we (13) and others (14) mapped the Acact gene to chromosome 1 near the ald locus. These observations prompted us to ask whether ald results from an Acact mutation.
In the current study, we examined whether ald and Acact are allelic through genetic crosses of AKR and AcactϪ/Ϫ mice and by molecular analysis of the Acact mRNA, protein, and enzyme activity levels in AKR mice. The results indicate that ald is indeed due to a naturally occurring mutant Acact allele but that the ald protein is capable of catalyzing cholesterol esterification normally. This finding suggests that the expression of the adrenocortical cholesterol ester depletion phenotype in AKR mice is caused by an altered susceptibility of the mutant protein to modifying influences.

MATERIALS AND METHODS
Mice-All inbred strains including AKR were obtained from The Jackson Laboratory (Bar Harbor, ME). AcactϪ/Ϫ mice (9) were of mixed C57BL/6 and 129/Sv genetic background and were genotyped with the polymerase chain reaction (PCR) as described (9). Mice were housed in a pathogen-free barrier facility operating on a 12-h light/12-h dark cycle and were fed rodent chow (Ralston Purina, St. Louis, MO).
Genetic Mapping of ald and Acact-Colocalization of ald and Acact was confirmed by typing microsatellite markers flanking a small region of chromosome 1 presumed to contain both loci. For Acact, a ((C57BL/ 6J ϫ Mus spretus)F1 ϫ C57BL/6J) interspecific backcross mapping panel (15) was typed, and for ald, a set of (AKXL)RI strains was typed. RI strain and progenitor control DNAs were purchased from The Jackson Laboratory. Genomic DNA was amplified with PCR primers for microsatellite markers (Research Genetics, Huntsville, AL) as described (16). Amplification products were separated on 5% polyacrylamide gels and detected by autoradiography. Variant alleles were scored, and the segregation patterns were entered into a Map Manager version 2.6.5 (17) data base containing either backcross (13) or RI strain data. The backcross panel has been typed for ϳ400 markers and the AKXL strains for 343 markers (17) distributed throughout the genome. Map positions were determined by minimizing the number of double and multiple recombination events. These mapping data have been deposited in the Mouse Genome Data Base (accession number MGD-JNUM-42073).
Tissue Lipid Analysis-For neutral lipid staining, adrenal glands were perfusion fixed in 3% paraformaldehyde in Tyrode's solution, dissected from surrounding tissues, and frozen. Vibratome-cut sections were subsequently stained with Oil Red O (18). For chromatographic analysis, lipids were extracted from adrenal gland homogenates with the Bligh-Dyer method (19), and high-performance thin-layer chromatography (HPTLC) was performed as described (20).
Immunoblot Analysis-Tissues were homogenized in 20 mM Tris, 150 mM NaCl, and 0.1% Triton X-100 (pH 7.4). An equal volume of homogenization buffer containing 10% SDS was then added to protein aliquots (final concentration, 5% SDS); the samples were heated to 65°C for 20 min, and protein aliquots were size fractionated by SDS-polyacrylamide gel electrophoresis with 10% gels. The separated proteins were transferred to nylon membranes and immunoblotted with a rabbit antiserum generated against a glutathione S-transferase/mouse ACAT fusion protein containing the amino-terminal 120 residues of the mouse ACAT protein (21). The low density lipoprotein receptor-related protein was detected with a rabbit antiserum (22). Binding of the antibodies was detected with an enhanced chemiluminescence kit (Amersham Corp.).
Cholesterol Esterification Assays-Adrenal glands were homogenized in 0.1 M sucrose, 50 mM KCl, 40 mM KH 2 PO 4 , and 30 mM EDTA (pH 7.4) with a Potter-Elvehjem homogenizer. After removal of intact cells and nuclei by centrifugation of homogenates at 600 ϫ g for 10 min, total membranes were isolated by ultracentrifugation (100,000 ϫ g pellet).
The rate of incorporation of [ 14 C]oleoyl-CoA (Amersham Corp.) into cholesterol esters was assayed essentially as described by Erickson et al. (25). Reactions were performed at 37°C for 5 min with 100 -150 g of protein homogenate and 25 M oleoyl-CoA (specific activity, ϳ18 mCi/mmol). Exogenous cholesterol (20 nmol) was added to the reaction by preincubating membranes for 30 min at 37°C with phosphatidylcholine:cholesterol (4:1 molar ratio) liposomes. In some experiments, exogenous cholesterol was provided as an acetone solution.
Transient Transfection of ACAT-deficient Chinese Hamster Ovary Cells-ACAT-deficient Chinese hamster ovary (CHO)-(AC29) cells (provided by Dr. T. Y. Chang, Dartmouth University, Hanover, NH) were transfected with pcDNA3 mammalian expression vectors (Invitrogen, Carlsbad, CA) containing the wild-type human, wild-type mouse, or AKR mouse (ald) ACAT cDNAs. DNA vectors (1 g) were transfected with LipofectAMINE (Life Technologies, Inc.) into equivalent amounts of cells (4 ϫ 10 5 ) plated on 6-well dishes. Approximately 48 h after transfection, cells were washed twice with phosphate-buffered saline, harvested by scraping, and homogenized in the buffer described above using a syringe and 10 passes through a 25-gauge needle. Total membranes (excluding nuclei) were isolated, and immunoblotting and cholesterol esterification assays were performed as described above.

RESULTS
We previously mapped Acact to a region of mouse chromosome 1 (13) containing the ald locus (8). Because the phenotype of AcactϪ/Ϫ mice included cholesterol ester depletion in the adrenal cortex of both male and female mice (9), we performed refined mapping of Acact and ald with microsatellite markers flanking the region containing both loci, utilizing DNA from backcrossed or recombinant inbred strains. Acact and ald colocalized within the proximal to distal interval D1Mit346-At3 (Fig. 1). The segregation pattern of Acact exhibited no recombination with D1Mit14 or D1Mit33 among 62 or 55 backcross progeny, respectively. ald was localized proximal to D1Mit33 but within the 95% confidence interval for the location of Acact, indicating that the Acact and ald loci were indistinguishable as determined within the resolution of these genetic markers.
To determine whether Acact and ald were allelic, we performed two genetic experiments designed to test whether the ald locus would complement the AcactϪ locus. We crossed AcactϪ/Ϫ mice (mixed C57BL/6 and 129/Sv background) with ald/ald (AKR) mice and assessed the adrenal neutral lipid content in Oil Red O-stained adrenal sections of male postpubertal F 1 offspring. Whereas Acactϩ/ϩ and Acactϩ/Ϫ control male mice had abundant neutral lipids in their adrenal cortex, male postpubertal F 1 offspring (carrying one ald and one AcactϪ allele) exhibited marked adrenocortical lipid depletion, similar to that in AcactϪ/Ϫ controls ( Fig. 2A). In a separate experiment, we crossed Acactϩ/Ϫ mice with ald/ald mice and analyzed the adrenal gland cholesterol ester content of the postpubertal male F 1 offspring by HPTLC. All F 1 offspring that carried an AcactϪ allele and an ald allele had marked cholesterol ester depletion (Fig. 2B), similar to that observed in AcactϪ/Ϫ controls and less than that observed in ald/ald controls. In contrast, control littermate F 1 offspring carrying an Acactϩ allele and an ald allele had cholesterol ester levels equivalent to or greater than that in Acactϩ/Ϫ controls ( Fig.   FIG. 1. Colocalization of ald and Acact loci demonstrated by refined genetic mapping with microsatellite markers. Microsatellite markers flanking a small region of chromosome 1 presumed to contain the Acact and ald loci were typed as described under "Materials and Methods." A ((C57BL/6J ϫ Mus spretus)F1 ϫ C57BL/6J) interspecific backcross mapping panel (15) was typed for Acact mapping, and a set of (AKXL)RI strains was used for ald mapping. Recombination frequencies and genetic distances (centimorgan Ϯ S.E.) are indicated between loci. 2B). The failure of the ald allele to complement the AcactϪ allele in both experiments indicated that the two loci were allelic.
Immunoblotting of preputial gland homogenates from adult male mice of 10 inbred strains demonstrated that the Acact protein from AKR mice migrated at a lower molecular mass (ϳ42 kDa) on SDS-PAGE than the ϳ45-kDa protein in the other mouse strains (Fig. 3A). Similarly, a protein of reduced molecular mass was observed in adrenal gland homogenates from male and female AKR mice and in ovary homogenates from female AKR mice (not shown). Quantitative analysis of the Acact protein in adrenal homogenates of AKR mice demonstrated that its levels in males and females were reduced to ϳ33 and ϳ46%, respectively, of the levels in male control (C57BL/6 ϫ 129/Sv) mice (Fig. 3B).
Northern blotting of RNA from the preputial glands of adult AKR mice revealed that the Acact mRNA was less abundant and slightly smaller than that of control mice (Fig. 4A). Similar findings were observed in RNA samples from kidney and testes of AKR mice (data not shown). Because the immunoblotting and Northern blotting experiments suggested that the ald protein may be truncated, we examined the cDNA sequences of the Acact mRNA and observed several differences as compared with the wild-type Acact cDNA (14). Initial 5Ј-RACE experiments followed by cDNA sequencing demonstrated that the ald cDNA contained a 118-bp deletion spanning part of the 5Ј untranslated region and the initial coding sequences (cDNA nucleotides 790 -897 (14)). To further investigate this finding, we performed reverse transcriptase-PCR experiments using primers located on either side of the deleted region (Fig. 4B). Whereas a 312-bp wild-type product was consistently amplified from cDNA of control mice, a 194-bp product (reflecting the 118-bp deletion) was amplified from all tissues tested of both male and female AKR mice; the 194-bp product was also observed at very low levels in the cDNA from the control mice. DNA sequencing of this amplification product demonstrated that it lacked the identical 118 bp identified in the 5Ј-RACE For A, AKR (ald/ald) mice were crossed with AcactϪ/Ϫ mice, and the lipid content of adrenal glands of the F 1 male postpubertal offspring was analyzed by Oil Red O staining. For B, AKR (ald/ald) mice were crossed with Acactϩ/Ϫ mice, and adrenal lipid extracts of male postpubertal F 1 offspring were analyzed by HPTLC. All F 1 offspring mice were genotyped for the ϩ or Ϫ allele at the Acact locus as described (9). Oil Red O staining of frozen sections and HPTLC analysis of lipid extracts were as described under "Materials and Methods."

FIG. 3. Mutant ACAT protein in AKR mice demonstrated by immunoblotting.
A, immunoblots of preputial gland homogenates from 10 different inbred strains of mice demonstrating an ACAT protein of reduced molecular mass specific for the AKR strain. Whole-tissue homogenates from postpubertal male mice were prepared, and protein samples (75 g) were analyzed by immunoblotting with an antiserum that recognizes the mouse Acact protein (21). B, immunoblots of adrenal homogenates demonstrating that the ACAT protein is reduced in molecular mass and detected in lower amounts in both female and male AKR mice. Trace amounts of a protein migrating identically to the reduced mass protein were sometimes observed in homogenates from wild-type mice. Indicated amounts of protein samples from whole cell homogenates of adult mice were analyzed by immunoblotting for Acact as described above. Sample aliquots were also immunoblotted with an antiserum that recognizes the low density lipoprotein receptor-related protein (LRP) (22) as an internal standard. Densitometric scanning of the autoradiogram and comparison of ACAT and LRP bands revealed that the mutant ACAT protein in male and female AKR mice was detected at ϳ33 and ϳ46%, respectively, of the levels in control male wild-type mice (C57BL/6 ϫ 129/Sv). experiments. DNA sequencing from C57BL/6 genomic DNA revealed that the 118 bp deleted in the ald cDNA corresponded to an exon containing the AUG translation initiation codon. In addition to the deleted sequences, the ald cDNA contained two missense mutations: A 3 G at cDNA nucleotide 1248, resulting in Ile 3 Val at amino acid 147; and C 3 T at nucleotide 1422, resulting in His 3 Tyr at amino acid 205. Several other nucleotide differences were observed (A 3 G at position 917, A 3 G at 971, and A 3 C at 1232) that did not result in amino acid coding differences.
To determine whether the protein product of the ald allele could esterify cholesterol, we performed cholesterol esterification activity assays on adrenal membranes harvested from AKR mice and controls. The adrenal membranes from adult male AKR mice had activity levels (121 Ϯ 11 pmol/mg protein/ min) similar to those of membranes from wild-type control (C57BL/6 ϫ 129/Sv) male mice (111 Ϯ 17 pmol/mg protein/min) and much greater than those of AcactϪ/Ϫ controls (3.8 Ϯ 0.5 pmol/mg protein/min) (Fig. 5A). Membranes from female AKR mice exhibited ϳ3-4-fold greater activity (351 Ϯ 171 pmol/mg protein/min) than those from male mice (consistent with the higher cholesterol ester in adrenal glands of female mice (26)). Because these assays were performed at V max , with saturating concentrations of cholesterol substrate (20 nmol), we hypothesized that the activity levels of the ald protein might be altered at lower substrate concentrations owing to altered affinity of the protein for cholesterol substrate. We therefore analyzed activity levels of postpubertal male AKR adrenal membranes in response to various concentrations of cholesterol. At each cholesterol concentration tested, however, the cholesterol esterification activity in membranes from AKR mice was equal to or greater than that of wild-type control membranes (Fig. 5B).
To confirm that the protein expressed by the ald allele could catalyze cholesterol esterification, we transiently expressed ald and wild-type Acact cDNAs in cultured ACAT-deficient (AC29) CHO cells (27) and measured cholesterol esterification activity in membrane homogenates. The transfection of the ald cDNA resulted in the expression of an Acact protein of lower molecular mass than that obtained with wild-type cDNAs, confirming the results of the immunoblot experiments in tissues from AKR mice. Membranes prepared from cells transfected with ald cDNA catalyzed cholesterol esterification at levels much greater than membranes of untransfected cells and similar to that of membranes from cells transfected with wild-type cDNA (Fig. 6). Normal cholesterol esterification activity was also observed in a pulse cholesterol esterification assay performed on intact transfected AC29 cells (data not shown). DISCUSSION ald, a naturally occurring mutation in the AKR inbred mouse strain, is associated with cholesterol ester depletion in the adrenal cortex, which becomes manifest at the time of sexual maturation (3). In the current study, we provide genetic and molecular evidence indicating that ald is associated with a naturally occurring mutant Acact allele. The AKR strain carries mutations at the Acact locus that result in an aminoterminal deletion in the Acact protein and two amino acid changes compared with the wild-type protein. Despite these differences, the ACAT protein from the ald allele catalyzed cholesterol esterification at levels similar to that of the wildtype protein. This finding suggests that the adrenocortical cholesterol ester depletion phenotype due to the ald mutation results from an altered susceptibility of the mutant ald protein to modifying influences present in vivo. FIG. 4. Mutant Acact mRNA in AKR mice. A, Northern blot demonstrating that the Acact mRNA is reduced in size and amount compared with that in C57BL/6 ϫ 129/Sv wild-type mice. RNA was isolated from preputial glands and subjected to Northern blotting with a 501-bp cDNA probe from the 5Ј coding sequences. The autoradiogram was exposed for 1 week. B, reverse transcriptase-PCR demonstrating a deletion in the Acact mRNA in tissues from male or female AKR mice. cDNA was prepared from RNA isolated from tissues as described under "Materials and Methods." Primers (triangles) flanking the first coding exon were used to amplify a fragment from the 5Ј region of the cDNA. The wildtype allele results in a 312-bp amplification product. The ald allele yields a 194-bp product. Sequencing of this PCR product revealed that it lacked the first coding exon sequences. The asterisks indicate two other transitions between the AKR and B6/129 Acact alleles that result in amino acid differences (A 3 G at cDNA nucleotide 1248, resulting in Ile 3 Val at amino acid 147, and C 3 T at nucleotide 1422, resulting in His 3 Tyr at amino acid 205). UT, untranslated.
Two lines of genetic evidence from our studies indicate that ald and Acact are allelic. First, refined genetic mapping demonstrated colocalization of the two loci to the same region of chromosome 1. Second, a cross between AcactϪ/Ϫ and AKR (ald/ald) mice demonstrated a lack of complementation between the ald and AcactϪ alleles with regard to adrenocortical lipid depletion in male postpubertal mice. Male postpubertal F 1 offspring from this cross with an ald/Ϫ genotype were characterized by cholesterol ester depletion in the adrenal cortex. If ald and Acact were alleles of different loci, then this cross would result in heterozygosity at each locus (i.e. Acactϩ/Ϫ and ald/ϩ), and male postpubertal offspring would be expected to have abundant cholesterol esters in the adrenal cortex at levels similar those in Acactϩ/Ϫ mice or ϩ/ald mice. The fact that DNA from AKR mice could not correct the adrenocortical lipid depletion caused by the AcactϪ allele provides near-definitive evidence that the ald mutation results from a mutant Acact allele.
The RNA and immunoblot analyses demonstrate clearly that the ald allele results in the production of a mutant Acact protein. Of 10 inbred strains surveyed, an Acact protein of lower molecular mass was found only in the AKR strain. The analysis of Acact cDNA from AKR mice demonstrated that the truncated Acact protein lacks the first coding exon containing the AUG translation initiation codon. Further experiments will be necessary to identify the genomic mutation that leads to the missing exon. The lack of this exon in the ald mRNA results in an amino-terminal deletion of at least the first 30 amino acids of the Acact protein. We hypothesize that translation of the Acact protein from the ald mRNA begins at the AUG codon at amino acid 34; this AUG is in the correct reading frame and is located in a Kozak consensus sequence for translation initiation (28). Supporting this hypothesis, the expression of the ald cDNA in cultured cells resulted in a truncated protein of identical molecular mass to that observed in tissues from AKR mice. The mutant protein was detected at lower levels than the wild-type Acact protein by immunoblotting, both in homogenates from male and female AKR mice and in cultured cells expressing equal amounts of wild-type or mutant Acact cDNAs. These immunoblot results may truly reflect lower levels of the ald protein, possibly due to a lower rate of translation originating from the alternate AUG start codon. However, it is also possible that the amino-terminal deletion in the ald protein resulted in altered reactivity of this truncated protein with the FIG. 5. Normal cholesterol esterification activity in adrenal gland membranes from AKR mice. A, cholesterol esterification activity of adrenal membranes from adult male and female AKR mice compared with male Acactϩ/ϩ and AcactϪ/Ϫ mice. Adrenal membranes were prepared as described under "Materials and Methods," and samples (200 g) were assayed for cholesterol esterification activity. Exogenous cholesterol substrate was provided as cholesterol/phospholipid liposomes. Values are the mean Ϯ SD for two samples of adrenal glands pooled from four mice each and assayed in duplicate. B, cholesterol esterification activity of male AKR mice in response to different concentrations of cholesterol substrate. Cholesterol esterification assays were performed as described above, except that the cholesterol was added as an acetone solution. Each data point is the mean of pooled adrenal glands from four mice in duplicate experiments. Approximately 48 h after the transfection, cells were harvested, and the membranes were prepared and assayed for cholesterol esterification activity as described under "Materials and Methods." Data are shown for duplicate samples from a single transfection experiment; the experiment was repeated once, and the identical result was obtained. The same membrane samples (75 g) were immunoblotted with an antiserum that recognizes Acact (21) to demonstrate protein expression (inset). polyclonal antiserum (which recognizes epitopes at the amino terminus of ACAT) and that the immunoblotting data reflect this difference.
Despite the alterations, the ald protein catalyzed cholesterol esterification at levels equivalent to or greater than those of the wild-type Acact protein. This finding was observed both in adrenal tissues from adult male and female postpubertal AKR mice and in cultured cells expressing the mutant protein. Similar findings indicating that the ald protein can catalyze cholesterol esterification have been observed in pulse assays of cultured peritoneal macrophages from AKR mice. 2 We observed normal or supranormal activity levels in assays carried out both at V max and at a range of cholesterol substrate concentrations. Thus, it is clear that the ald protein can catalyze cholesterol esterification, at least under the conditions utilized in our experiments. These data provide important structurefunction information indicating that the Acact protein can still catalyze cholesterol esterification at normal levels despite an amino-terminal truncation (likely of 33 amino acids) and the amino acid changes at residues 147 and 205. Although this finding was somewhat surprising given the cholesterol ester depletion phenotype, it was not totally unexpected, inasmuch as the adrenal glands of AKR mice contain normal levels of cholesterol esters before sexual maturation. It is possible that the ald protein is associated with reduced ACAT activity in intact male AKR adrenal glands and that we were unable to reproduce the conditions or modifying factors that inhibit the activity in adrenal glands in our in vitro assays.
How does the ald allele contribute to cholesterol ester depletion at the time of puberty? The answers to this question are largely speculative. It is clear that alterations of different proteins involved in cholesterol metabolism can cause adrenocortical lipid depletion. Phenotypes of adrenocortical lipid depletion have been observed for knockouts of Acact (9) and Apoa1 (26) and in the naturally occurring ald mutations in AKR and DBA/2 (6) mice. In contrast to the cholesterol ester depletion in both male and female AcactϪ/Ϫ and Apoa1Ϫ/Ϫ mice, however, the ald phenotype in AKR and DBA/2 mice is conditional and is manifested only in the presence of modifying influences, such as androgen production at the time of puberty. Supporting the hypothesis that adrenocortical lipid depletion can result from the interactions of different factors, preliminary studies in DBA/2 mice suggest that the primary defect is not due to the Acact locus and that at least two loci are responsible for the phenotype. 3 In AKR mice, we speculate that an increase in androgens affects or interacts with the ability of the ald protein to esterify cholesterol or to facilitate the storage of cholesterol esters. One possibility is that the amino-terminal deletion in the ald protein affects protein-protein interactions or the localization of the ald protein in intracellular compartments or in cell membranes and that these alterations influence the susceptibility of the protein to androgen-associated factors. Identifying factors that modulate the adrenocortical lipid depletion phenotype could provide a better understanding of cholesterol metabolism in adrenocortical cells and intracellular cholesterol metabolic pathways.