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Endocrinology 143(3):801– 806
Copyright © 2002 by The Endocrine Society

Adrenal Neutral Cholesteryl Ester Hydrolase:
Identification, Subcellular Distribution, andSex Differences
FREDRIC B. KRAEMER, WEN-JUN SHEN, VANITA NATU, SHAILJA PATEL, JUN-ICHI OSUGA,SHUN ISHIBASHI, AND SALMAN AZHAR
Veterans Affairs Palo Alto Health Care System (F.B.K., S.A.), Palo Alto, California 94304; Department of Medicine (F.B.K.,
W.-J.S., V.N., S.P.), Stanford University, Stanford, California 94305; Department of Metabolic Disease (J.-i.O.), Universityof Tokyo, Tokyo 113, Japan; and Department of Internal Medicine (S.I.), Jichi Medical School, Tochigi, Japan
Adrenals express a high level of neutral cholesteryl ester hydrolase (CEH) activity, and male rats have greater activity
than females; however, the identity of the enzyme(s) responsible for this activity and the basis for the sex differences are
unknown. Using mice in which hormone-sensitive lipase(HSL) was inactivated by homologous recombination (HSLⴚ/ⴚ), neutral CEH activity was reduced more than 98% compared with controls. Female HSL ⴚ/ⴚ mice showed a reduction in stimulated corticosterone values. Mechanical separation of rat adrenals revealed less HSL in the outer than the
inner cortex. Examination of subfractions of rat adrenals
showed that immunoreactive HSL was prominently expressedin microsomes, with lesser amounts in the cytosol and little tono HSL in mitochondrial and nuclear fractions or the lipid
droplet. Four- to 10-fold more neutral CEH activity was in the
microsomal fraction than any other fraction. No sex differences in the expression or subcellular distribution of HSL
protein were found; however, neutral CEH activity was lower
in the microsomal fraction of females, and female adrenals
contained more cholesteryl esters. Thus, HSL appears to be
responsible for most, if not all, of adrenal neutral CEH activity, is prominently expressed in microsomes, and its activity
is influenced by sex. (Endocrinology 143: 801– 806, 2002)

M

OST CELLS, INCLUDING adipose tissue, adrenals,
ovaries, testes, and placenta, have been shown topossess a neutral cholesteryl ester hydrolase (CEH) (1– 4).
Steroidogenic cells are particularly enriched in this activity(5–10). The presence of substantial pools of cholesteryl esters
in the adrenal cortex of many species, including rat andhuman, and the recognition that this potential substrate enters the steroidogenic pathway as free cholesterol, signifies
the important role of cholesterol esterase in the regulation ofsteroidogenesis (11–15). The neutral CEH first described by
Dailey et al. (16) in canine adrenal homogenates, and later
demonstrated in other steroidogenic tissues (11, 13, 14), isdistinct from that of lysosomal (acid) cholesterol esterase.
Although steroidogenic tissues contain a lysosomal cholesterol esterase, it constitutes only a small percentage of total
CEH activity (⬍5% in the adrenal). Several lipases have been
identified that possess neutral CEH activity; for instance,neutral cholesterol esterase activity in liver appears to be due
to a bile salt-stimulated enzyme that is identical to pancreaticcholesterol esterase (17, 18) and to a unique hepatic neutralcholesteryl ester hydrolase (19 –21). Immunological techniques have differentiated adrenal CEH from the pancreatic/
intestinal cholesterol esterase, which has a lower optimal pH(6.6) than neutral CEH (7.0 –7.5), an absolute requirement forbile acids, and catalyzes both cholesteryl ester formation andhydrolysis (14, 22). The neutral cholesterol esterase activity
purified from the adrenal has been shown to be identical tohormone-sensitive lipase (HSL) purified from adipose tissue(5). Moreover, immunoreactive HSL of approximately 84
kDa (7, 23) and HSL mRNA of 3.3 kb (3, 24) can be detectedin adrenal, just as in adipose tissue.
The intracellular distribution of neutral CEH is somewhat
uncertain. Approximately 70% of adrenal neutral CEH has
been reported to be associated with the cytosol (8, 25); however, the soluble CEH could be resolved into two components, one of which was associated with lipid (25). It is,therefore, unclear whether the appearance of CEH in thecytosol reflects its true locus in intact cells or is a procedural
artifact whereby activity loosely associated with lipid droplets or membrane structures is easily released.
Male rats have greater than 2-fold the adrenal CEH specificactivity of females (26), although steroid output is lower inmale rats (14). Following gonadectomy, the levels of adrenal
CEH in males is unaltered, whereas in females it tends toincrease (26). In the current studies, we have assessed thesubcellular distribution of HSL in the rat adrenal and havereexamined the effects of sex on neutral CEH expression in
the adrenal in rats.
Materials and Methods
Chemicals
Reagents were obtained from the following sources: BSA (fraction V)(Intergen Co., Purchase, NY); sodium taurocholate, l-␣ phosphatidylcholine, Triton X-100, cholesterol oleate (Sigma, St. Louis, MO); cholesterol[1-14C]oleate (E. I. Dupont de Nemours and Co., Boston, MA);
chloroform, methanol, heptane (J. T. Baker, Inc., Phillipsburg, NJ). Allother chemicals were obtained from standard commercial sources.

Abbreviations: CEH, Cholesteryl ester hydrolase; HSL, hormonesensitive lipase.

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Endocrinology, March 2002, 143(3):801– 806

Animals
Male and female Sprague Dawley rats (Harlan, Indianapolis, IN)
were obtained and maintained on ad libitum rat chow and tap water witha 12-h light, 12-h dark cycle in accord with accepted standards of humane
animal care outlined in Stanford University guidelines. Experiments
were performed with tissues obtained from adult rats (180 –240 g). HSL⫺/⫺ mice were generated as described previously (27). Homozygous
HSL ⫺/⫺ mice were identified from heterozygous crosses by Southernblot of EcoRI-digested tail DNA as described previously (27). In someexperiments, mice were injected sc with ACTH (25 ␮g) and serum
obtained 60 min later for measurement of corticosterone. Animals were
killed by CO2 asphyxia, and adrenals were immediately removed,
washed three times with ice-cold PBS, and processed immediately.

Cell fractionation
Adrenals were subfractionated by differential centrifugation in sucrose. Briefly, adrenals from three animals were homogenized in 2 mlof Buffer A [0.25 m sucrose, 1 mm EDTA, 10 mm Tris-HCl (pH 7.4), and2 ␮g/ml leupeptin] and filtered through mesh cloth. The homogenate
was centrifuged at 600 ⫻ g for 10 min. The pellet was washed once with
2 ml of Buffer A, resedimented, and used as the nuclear-cell debris
fraction. The 600 ⫻ g supernatant was centrifuged at 14,000 ⫻ g for 15
min; the resulting pellet was resuspended in 2 ml Buffer A, centrifuged
at 8,500 ⫻ g for 15 min, and the sediment used as the mitochondrial
fraction. The 14,000 ⫻ g supernatant was centrifuged at 100,000 ⫻ g for
60 min, with the resulting pellet identified as the microsomal fraction
and the supernatant as the cytosolic fraction. In some experiments, thezona glomerulosa was separated from the remainder of the cortex andthe medulla by removing the capsule and adhering tissue under adissecting microscope (28).

Neutral CEH activity
Neutral CEH activity was measured as previously described (23).
Briefly, a micellar substrate was prepared by adding cholesterol[114C]oleate (2.5 ␮Ci) to a chloroform solution containing 5.209 mg unlabeled cholesterol oleate and 24.32 mg phosphatidylcholine. The solvent was removed with a stream of N2 in a 37-C water bath, and thelipids suspended in 8.0 ml of 100-mm KPO4 buffer (pH 7.0) containing
8.6 mg Na taurocholate. The suspension was transferred to a waterjacketed glass vessel maintained at 4 C and sonicated for 3 h with aBranson Sonifier/Cell Disruptor model W-350 on an output setting of
5.0 (50%). The substrate was centrifuged at 3000 rpm for 10 min toremove metallic fragments released by the probe, and stored under
nitrogen at 4 C for up to 1 wk. Aliquots of extracts were mixed with
substrate and incubated at 37 C for varying times up to 1 h. The reactions
were stopped by addition of chloroform:methanol:heptane (250:230:
180). After the addition of borate/carbonate buffer (0.1 m, pH 10.5), thetubes were vortexed, centrifuged, and aliquots of the upper phase taken
for liquid scintillation counting in a Beckman Coulter, Inc. (Fullerton,CA) scintillation counter. The results are expressed in nmol of cholesteryl oleate hydrolyzed/mg protein/h.

Immunoblotting
Samples were suspended in 50 mm Tris, pH 8.0; 1 mm EDTA; 1 mm
phenylmethylsulfonyl fluoride; 1 U/ml leupeptin; and 0.2 mg/ml aprotinin, electrophoresed on 10% polyacrylamide gels under reducing conditions, transferred to nitrocellulose, incubated with antirat HSL/fusion
protein IgG, and visualized by chemiluminescence as described previously (23).

Analytical proceduresProtein was measured with a bicinchoninic acid protein assay kit(Pierce Chemical Co., Rockford, IL). Cellular cholesteryl ester mass wasmeasured as described previously (29). Serum corticosterone was measured by RIA (30).

Statistics
Data are expressed as mean ⫾ sem. Statistical analyses were performed by ANOVA and comparisons among groups by Bonferroni/

Kraemer et al. • Adrenal HSL

Dunn using GB-STAT software (Dynamic Microsystems, Inc., Silver
Spring, MD) on a Power Macintosh computer.

Results

To explore the contribution of HSL to the total neutral CEH
activity in the adrenal, we examined neutral CEH activity inextracts of adrenals from normal and HSL ⫺/⫺ mice (27). As
shown in Table 1, normal wild-type male mice had higher
neutral CEH activity than female mice (P ⬍ 0.01). Importantly, inactivation of HSL by homologous recombination
resulted in the loss of more than 98% of neutral CEH activity
in both male and female mouse adrenals (P ⬍ 0.0001). Thus,
it appears that HSL is responsible for the vast majority, if notall, of the neutral CEH activity in the adrenal. To determine
whether the absence of adrenal neutral CEH activity influences adrenal steroidogenesis, the response of serum corticosterone to ACTH stimulation was examined in normal andHSL ⫺/⫺ mice (Table 2). Similar to previous observations inrats (14), female wild-type mice had higher serum corticosterone values than male wild-type normal mice (P ⬍ 0.05).
Interestingly, loss of neutral CEH activity by inactivation of
HSL resulted in a reduction in corticosterone response to
ACTH that was highly significant in female mice (P ⬍ 0.01)
but failed to reach statistical significance in male mice.
Because the adrenal contains both mineralocorticoid- andglucocorticoid-producing zones, it was of interest to determine the relative levels of HSL expression in the zona glomerulosa and other regions of the adrenal. To accomplish
this, the capsule (and adherent cells of the zona glomerulosa)of rat adrenals was mechanically separated from the innercortex and medulla and protein extracts immunoblotted for
HSL. As shown in Fig. 1, the relative levels of immunoreactive HSL were lower in extracts of the outer cortex (glomerulosa) than in the inner cortex.
Although neutral CEH activity has been biochemically
localized to cytosolic and particulate fractions of the adrenal,
it is not clear which, if any, intracellular membranes are
involved. To explore the intracellular localization of neutral
CEH further, adrenal homogenates were subfractionatedand the presence of HSL in the various fractions determinedby immunoblotting. As shown in Fig. 2, immunoreactive
HSL was prominently expressed in the microsomal fraction,with lesser amounts in the cytosolic fraction and little to no
HSL detected in mitochondrial and nuclear-cell debris fractions or the lipid droplet (P ⬍ 0.0001) when fractions were
TABLE 1. Neutral CEH activity in adrenals from normal wildtype and HSL⫺/⫺ mice
Activity
nmol/h䡠mg protein

NormalMale
Female1.658 ⫾ 0.141
0.962 ⫾ 0.19a

HSL⫺/⫺
Male
Female0.019 ⫾ 0.008b
0.015 ⫾ 0.004b

Adrenals were homogenized and assayed for neutral CEH activity
as described in Materials and Methods. The results represent themean ⫾ SEM of five normal and five HSL⫺/⫺ mice. a P ⬍ 0.01 compared with male wild-type normal; b P ⬍ 0.0001 compared with normal wild-type.

Kraemer et al. • Adrenal HSL

Endocrinology, March 2002, 143(3):801– 806 803

TABLE 2. ACTH-stimulated serum corticosterone values innormal wild-type and HSL⫺/⫺ mice
Corticosterone
ng/ml

NormalMale
Female352 ⫾ 25581 ⫾ 111a

HSL⫺/⫺
Male
Female285 ⫾ 30
247 ⫾ 23b

Mice were injected with ACTH (25 ␮g) and serum obtained 60 min
later for measurement of corticosterone. The results represent themean ⫾ SEM of 10 normal male, 6 normal female, 9 HSL⫺/⫺ male, and6 HSL⫺/⫺ female mice. a P ⬍ 0.05 compared with male wild-type
normal; b P ⬍ 0.01 compared with normal wild-type.

FIG. 1. HSL expression in the zona glomerulosa and the inner cortex
of rat adrenals. Adrenals were removed, rinsed in PBS, and the outer
cortex (zona glomerulosa) was separated from the remainder of thecortex and the medulla by removing the capsule and adhering tissue
under a dissecting microscope. The adrenal fractions were homogenized and aliquots were immunoblotted with antirat HSL/fusion protein IgG, as described in Materials and Methods.

loaded based on equivalent amounts of protein. The majorityof neutral CEH activity was detected in the microsomal fraction (Fig. 2B); however, there was a greater discrepancy in the
distribution of neutral CEH activity than immunoreactive
HSL among the fractions, with 4- to 10-fold more neutral
CEH activity found localized to the microsomal fraction thanany of the other fractions (P ⬍ 0.0001).
Because cell content of neutral CEH may be influenced bysex steroid hormones (26), we compared CEH activity and
HSL protein levels in adrenals from mature male and female
rats. Figure 3A shows the neutral CEH activity in microsomal
and cytosolic fractions in adrenals from male and female rats.
While the majority of neutral CEH activity was found in the
microsomal fraction in both male and female rats, activity
was 20% lower in the microsomal fraction of female compared with male rats (P ⬍ 0.01). Neutral CEH activity in thecytosolic fraction was similar in males and females. As observed with neutral CEH activity, more immunoreactive
HSL was found in the microsomal fraction than in the cytosolic fraction in both males and females, but the differences
between microsomal and cytosolic HSL protein were not as

FIG. 2. Neutral CEH expression in subcellular fractions of rat adrenals. Adrenals were homogenized and subcellular fractions isolatedby differential centrifugation as described in Materials and Methods.
Aliquots of each fraction (15 ␮g) were immunoblotted with antirat
HSL/fusion protein IgG (A and B) and assayed for neutral CEH activity (C), as described in Materials and Methods. The results represent four independent measurements. N, Nuclei-cell debris fraction; Mit, mitochondrial fraction; Mic, microsomal fraction; C,
cytosolic fraction; L, lipid fraction.

great as observed for neutral CEH activity. In contrast to
neutral CEH activity, the expression of immunoreactive HSL
was similar in males and females in both microsomal andcytosolic fractions.
To determine whether the differences in neutral CEH ac-

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Endocrinology, March 2002, 143(3):801– 806

Kraemer et al. • Adrenal HSL

FIG. 4. Adrenal cholesteryl ester and unesterified cholesterol content
in male and female rats. Adrenals from six adult male and six female
rats were assayed for cholesterol content. *, P ⬍ 0.005.

FIG. 3. Neutral CEH expression in adrenals from male and female
rats. Adrenals from five adult male and five female rats were subfractionated into microsomes (Micro) and cytosol and assayed forneutral CEH activity (A) and immunoreactive HSL (B), as describedin Fig. 2. The data are representative of three separate experiments.
*, P ⬍ 0.01.

tivity observed in adrenals of female and male rats had
physiological effects, the content of cholesteryl esters in adrenals was assessed (Fig. 4). Consistent with the reduced neutral CEH activity seen, adrenals from female rats had 50%
more cholesteryl esters than adrenals from male rats (P ⬍0.005). In contrast, the content of unesterified cholesterol was
similar in adrenals from males and females.
Discussion

While neutral CEH activity has been observed in adrenals
for many years (16), the identity of the enzyme(s) responsible
for this activity has remained controversial. Even thoughbiochemical characterization (5) and immunological evidence (7, 23) had suggested that HSL was present in theadrenal, the possible existence of other neutral CEHs remained unanswered. In the current studies we have used
HSL⫺/⫺ mice to assess the contribution of HSL to the total
neutral CEH activity found in the adrenals. In view of the factthat neutral CEH activity was reduced more than 98% in bothmale and female HSL⫺/⫺ mice compared with wild-type
control mice, it can be concluded that HSL is responsible forthe vast majority, if not all, of the neutral CEH activity in themouse adrenal. Indeed, it is likely that the small amount of
neutral CEH activity observed in adrenals of HSL⫺/⫺ miceis due to residual activity of lysosomal acid lipase that canstill be seen at neutral pH (31). This observation expands theprevious findings that neutral CEH activity is completely
absent in adipose tissue and testes from HSL⫺/⫺ mice (27)and supports the notion that HSL is the primary neutral CEH
in adipose and steroidogenic tissues.

While adrenal steroidogenesis is primarily controlled by
the ability of ACTH to regulate the expression of steroidogenic acute regulatory protein and enzymes of the steroidogenic pathway, an increased delivery of cholesterol to mitochondrial cytochrome P450scc is also needed (14, 32). The
cholesterol used for steroidogenesis is derived from a combination of sources (12): 1) the mobilization of stored cholesteryl esters via the actions of neutral CEH, 2) lipoproteinderived cholesteryl esters obtained by cellular uptake, and 3)
de novo cellular cholesterol synthesis. It is apparent that thesystems for cholesterol delivery for steroidogenesis are redundant. However, our current findings that the loss ofneutral CEH activity by inactivation of HSL resulted in a
reduction in corticosterone response to ACTH support thenotion that the actions of neutral CEH are involved in thedelivery of cholesterol for steroidogenesis. Additional experiments will be required to quantitate the contributions of
the various sources of cholesterol and to document the mechanisms through which HSL is involved.
Although HSL has been considered to be a cytosolic enzyme in the adrenal (8, 25), our current findings suggest that
there is substantial enrichment of HSL within microsomes.
The localization of HSL to microsomes is consistent with theconcept that HSL might be tethered to proteins locatedwithin microsomes under basal conditions, and that thisprevents the enzyme from having access to the cholesterylester droplet substrate located within the cytosol. Following
lipolytic stimulation, a conformational change that alters thisprotein-protein interaction might occur, allowing HSL tointeract (translocate) now with the lipid droplet. The translocation of HSL to the lipid droplet has been demonstrated
in adipocytes (33–36), making it likely that it also occurs in
the adrenal; however, this awaits definitive experimental
documentation. Moreover, a novel docking protein, lipotransin, which possesses intrinsic ATPase activity, has recently been identified to potentially direct HSL distributionin adipose cells (37). Whether lipotransin is expressed in theadrenal and its subcellular localization remain to be determined. The reasons for the differences in our observation ofsubstantial enrichment of HSL in microsomes comparedwith previous reports of neutral CEH activity (8, 25) are notentirely clear; however, our current results are based on
immunological evidence, as well as activity. Althoughthese previous investigators reported between 58 and 70% ofKraemer et al. • Adrenal HSL

neutral CEH activity in the cytosol, they noted appreciable
amounts in microsomes. We used similar centrifugation
techniques for subfractionation as used previously. However, because protease inhibitors, which were included in ourbuffers, were previously omitted, it is possible that substantial amounts of HSL might have been released from themicrosomal fraction into the cytosol during preparation.
Of particular interest is our finding that the apparent microsomal enrichment of HSL is exaggerated when the distribution of neutral CEH activity is examined. Thus, it appears that the hydrolytic activity of the enzyme found within
the cytosol is lower than the enzyme located in microsomes.
HSL hydrolytic activity can be controlled by phosphorylation; phosphorylation by PKA increases hydrolytic activity(38), while phosphorylation at other sites by kinases such as
glycogen synthase kinase-4 and Ca2⫹/calmodulin-dependent protein kinase II impair the phosphorylation by PKA(39). In addition to phosphorylation, HSL activity appears to
be regulated by oligomerization, with the dimeric enzyme
exhibiting markedly increased activity (40). Thus, it is possible that differences in either phosphorylation or dimerization of HSL could explain the discrepancy between HSL
protein expression and neutral CEH activity in the cytosol.
Although additional experiments are required to explore
this, it is intriguing to speculate that there is a greater proportion of monomeric HSL found in the cytosol.
Previous workers have documented that neutral CEH activity in the adrenal was higher in male rats compared withfemales (26). Our findings show that there are no differencesin the expression of HSL protein in the adrenals of male andfemale rats, nor are there any differences in the subcellular
distribution of HSL protein in the adrenals of male andfemale rats; HSL immunoreactive protein is enriched to asimilar degree within the microsomal fraction of both maleand female rats. However, we show that HSL activity (neutral CEH) in the adrenal is lower in female rats and that thisreduction in activity is confined to the microsomal fraction.
As discussed above, in view of the similar amounts of HSL
protein expressed, differences in neutral CEH activity could
possibly be explained by differences in the phosphorylation
state of HSL, with either a greater percentage of HSL being
activated by phosphorylation in males or a greater percentage of HSL in females being phosphorylated at sites thatprevent activation. Likewise, the degree of dimerization of
HSL could possibly explain the sex differences, with a greater
proportion of HSL in males being found as a dimer. Additional experiments will be required to address these possibilities. Nonetheless, the lower neutral CEH activity observed in microsomal fractions of female adrenals appears tocorrelate with the functional consequence of an increase incholesteryl ester storage.
In summary, based on mice in which HSL was inactivatedby homologous recombination, HSL appears to be responsible for the vast majority, if not all, of adrenal neutral CEH
activity. A potentially significant physiological role for adrenal HSL is supported by the observation that female HSL⫺/⫺ mice showed a reduction in stimulated corticosterone
values. Less HSL is expressed in the outer cortex (glomerulosa) than the inner cortex, and immunoreactive HSL isprominently expressed within microsomes, with lesser

Endocrinology, March 2002, 143(3):801– 806 805

amounts in the cytosol and little to no HSL detected in
mitochondrial and nuclear fractions or the lipid droplet.
There was 4- to 10-fold more neutral CEH activity localized
to the microsomal fraction than any of the other fractions. No
sex differences in the expression or subcellular distributionof HSL protein in rat adrenals were found; however, its
activity is influenced by sex since neutral CEH activity was
lower in the microsomal fraction of females, and female
adrenals contain more cholesteryl esters.
Acknowledgments
Received June 27, 2001. Accepted November 14, 2001.
Address all correspondence and requests for reprints to: Fredric B.
Kraemer, M.D., Division of Endocrinology, S-005, Stanford University,
Stanford, California 94305-5103. E-mail: fbk@stanford.edu.
This work was supported in part by the Research Service of the
Department of Veterans Affairs, and by Grants DK-46942 and DK-49705
from the NIH.

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