Targeted disruption of hormone-sensitive lipase
results in male sterility and adipocyte hypertrophy,
but not in obesity
Jun-ichi Osuga*, Shun Ishibashi*†, Teruaki Oka‡, Hiroaki Yagyu*, Ryuichi Tozawa*, Akihisa Fujimoto§,
Futoshi Shionoiri*, Naoya Yahagi*, Frederic B. Kraemer¶, Osamu Tsutsumi§, and Nobuhiro Yamada*储
*Departments of *Metabolic Diseases, ‡Pathology, and §Obstetrics and Gynecology, Faculty of Medicine, University of Tokyo, Hongo, Tokyo 113, Japan; and¶Division of Endocrinology, Department of Medicine, Stanford University, Veterans Affairs Palo Alto Health Care System, Palo Alto, CA 94304

Hormone-sensitive lipase (HSL) is known to mediate the hydrolysis
not only of triacylglycerol stored in adipose tissue but also ofcholesterol esters in the adrenals, ovaries, testes, and macrophages. To elucidate its precise role in the development of obesityand steroidogenesis, we generated HSL knockout mice by homologous recombination in embryonic stem cells. Mice homozygous
for the mutant HSL allele (HSLⴚ兾ⴚ) were superficially normal
except that the males were sterile because of oligospermia.
HSLⴚ兾ⴚ mice did not have hypogonadism or adrenal insufficiency.
Instead, the testes completely lacked neutral cholesterol esterhydrolase (NCEH) activities and contained increased amounts ofcholesterol ester. Many epithelial cells in the seminiferous tubules
were vacuolated. NCEH activities were completely absent fromboth brown adipose tissue (BAT) and white adipose tissue (WAT)
in HSLⴚ兾ⴚ mice. Consistently, adipocytes were significantly enlarged in the BAT (5-fold) and, to a lesser extent in the WAT (2-fold),
supporting the concept that the hydrolysis of triacylglycerol was,at least in part, impaired in HSLⴚ兾ⴚ mice. The BAT mass wasincreased by 1.65-fold, but the WAT mass remained unchanged.
Discrepancy of the size differences between cell and tissue suggests the heterogeneity of adipocytes. Despite these morphological changes, HSLⴚ兾ⴚ mice were neither obese nor cold sensitive.
Furthermore, WAT from HSLⴚ兾ⴚ mice retained 40% of triacylglycerol lipase activities compared with the wild-type WAT. In conclusion, HSL is required for spermatogenesis but is not the only
enzyme that mediates the hydrolysis of triacylglycerol stored inadipocytes.

Hormone-sensitive lipase (HSL) is a multifunctional enzymethat catalyzes the hydrolysis of triacylglycerol stored inadipose tissue and cholesterol esters in the adrenals, ovaries,
testes, and macrophages (1, 2). It is encoded by a gene locatedon chromosome 19, and lacks homology to members of thepancreatic lipase gene family such as lipoprotein lipase(LPL) (3).
The lipolytic activities of HSL are under acute neuronal andhormonal control. Catecholamines and other lipolytic hormones
stimulate these activities through the reversible phosphorylationof serine by cyclic adenosine 3⬘,5⬘-monophosphate (cAMP)dependent protein kinase (PKA) (4). Conversely, insulin, anantilipolytic hormone, suppresses its activities by preventing
phosphorylation (5). Therefore, the activation of this enzyme inadipose tissues is thought to account for the elevation of plasma
free fatty acid (FFA) levels in various conditions such as
starvation or diabetes mellitus. Since mice lacking ␤3-adrenergic
receptor (6), a dominant form of ␤-adrenergic receptor expressed in adipocytes, are obese and since mice lacking the RII␤subunit of PKA are lean because of increased PKA activity (7),
it is reasonable to speculate that HSL mediates the regulation of
adiposity by the adrenergic signaling pathway.
In addition to the triacylglycerol lipase activity, neutral cholesterol ester hydrolase (NCEH) activity in the adrenals andovaries is mediated by HSL (2, 8, 9), implicating its role insteroidogenesis. HSL is also expressed in testicular cells, including spermatids and spermatozoa (10–12), suggesting its involvement in sperm development and兾or function. Furthermore,
NCEH activities expressed in aorta and macrophage cell lines
have similar hormone sensitivity to triacylglycerol lipase activities in adipocytes (13, 14). In addition, HSL overexpression
stimulates NCEH activities in macrophages (15). Therefore, it is
widely accepted that HSL is responsible for the NCEH activities
in macrophages (16–18), although there are those who disagree(19). Paradoxically, however, transgenic mice overexpressing
HSL under the control of scavenger receptor A promoterenhancer are not protected against atherosclerosis (20).
In an attempt to elucidate the bona fide functions of HSL, we
generated HSL knockout mice (HSL⫺兾⫺). These animals were
not obese and were as cold sensitive as wild-type mice. Unexpectedly, HSL⫺兾⫺ male mice were sterile because of oligospermia. These results indicate the critical role of HSL in spermatogenesis and the redundancy of lipases that catalyze the hydrolysis of triacylglycerol in adipose tissue.
Methods
Generation of the HSL Knockout Mice. The targeting vector con-struct was generated by insertion of a 1.4-kb PstI fragment withsequences of intron 6 to intron 7 as a short arm and 12 kb of HSL
genomic sequences including exons 1 to 5 as a long arm into the3⬘ and 5⬘ sides of the pol2neobpA expression cassette, respectively (11). Two copies of the herpes simplex thymidine kinase(tk) gene were inserted in tandem at the 3⬘ end of the short arm.
The resultant construct was linearized by SalI digestion andelectroporated into JH-1 embryonic stem cells. Selection with
G418 and 1-(2-deoxy-2-f luoro- ␤ - D -arabinofuranosyl)-5iodouracil (FIAU) was performed as already described (21).
Twenty-five of 144 clones resistant to both G418 and FIAU
harbored the desired product of homologous recombination as
judged by Southern blotting, and they were injected into blastocysts to generate chimeras. Chimeric males born to three
independent embryonic stem cell clones were crossed with
C57BL兾6 females, and they gave the germ-line transmission.
Mice were maintained on a 12-h dark兾light cycle and were fed
a normal chow diet. Unless otherwise indicated, tissues wereprepared from males between 9 and 14 weeks old.
Abbreviations: HSL, hormone-sensitive lipase; LPL, lipoprotein lipase; FFA, free fatty acid;
NCEH, neutral cholesterol ester hydrolase; BAT, brown adipose tissue; WAT, white adipose
tissue.
See commentary on page 535.
†To whom reprint requests should be addressed. E-mail: ishibash-tky@umin.ac.jp.
储Present address: Metabolism, Endocrinology and Atherosclerosis, Institute of Clinical

Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan.
The publication costs of this article were defrayed in part by page charge payment. This
article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.
§1734 solely to indicate this fact.

PNAS 兩 January 18, 2000 兩 vol. 97 兩 no. 2 兩 787–792

MEDICAL SCIENCES

Communicated by Richard J. Havel, University of California, San Francisco, CA, November 4, 1999 (received for review May 10, 1999)

Peritoneal Macrophages. One milliliter of 5% thioglycolate broth

was injected into the peritoneal cavities of mice. After 4 days, theperitoneal cavities were lavaged with 16 ml of ice-cold saline.
The cells were washed three times with PBS and resuspended in
DMEM to give a concentration of 106 cells per ml, and 10 ml per
dish was plated in 10-cm dishes. After incubation at 37°C for 2 h,
the nonadherent cells were removed by washing three times withwarmed PBS. Cells were incubated in DMEM containing 10%
FCS for 24 h and harvested for the experiments.
Northern Blot Analysis. Ten micrograms of total RNA was subjected to 1% agarose gel electrophoresis in the presence offormalin. The fractionated RNA was transferred to Hybond N(Amersham Pharmacia). The filters were hybridized to 32Plabeled cDNA probes corresponding to exon 1 or 8 sequences.
Immunoblot Analysis. Tissues were homogenized in buffer A (0.25
M sucrose兾1 mM EDTA兾2 ␮g/ml leupeptin兾50 mM Tris䡠HCl,
pH 7.0) and centrifuged at 100,000 ⫻ g for 45 min at 4°C. The
supernatant was used for the immunoblot analysis, which used anenhanced chemiluminescence kit (ECL; Amersham) and rabbit
anti-rat HSL antibody (12).
Assays for NCEH and Triacylglycerol Lipase Activity. Tissues were

homogenized in buffer A and centrifuged at 100,000 ⫻ g for 45
min at 4°C. The supernatant was used for the enzyme assay.
Macrophages were sonicated in buffer A and used directly forthe enzyme assay. NCEH activity was measured essentially as
described by Hajjar et al. (13), using a reaction mixture containing 6.14 ␮M cholesterol [1-14C]oleate (48.8 ␮Ci兾␮mol; 1 ␮Ci ⫽37 kBq). Triacylglycerol lipase activity was measured according
to a modified method of Hajjar et al. (13). In brief, the samples
were incubated at 37°C for 30 min in a final volume of 200 ␮l ofa reaction mixture containing 105 ␮M tri[3H]oleoylglycerol (99.4
␮Ci兾␮mol), 23.7 ␮M lecithin, 12.5 ␮M sodium taurocholate, 1 M
NaCl, and 85 mM potassium phosphate, pH 7.0. The highconcentration of NaCl was included to inactivate LPL, which isa major triacylglycerol lipase expressed by adipocytes.
Lipids and Hormones. Cholesterol, triacylglycerol, and FFA were

determined enzymatically with Determiner TC555, Determiner
TG555, and nonesterified fatty acids (NEFA) (Kyowa Medics,
Tokyo), respectively. Plasma glycerol levels were determined by
Determiner TG555, and glycerol in the incubation media wasdetermined by a radiometric assay according to Bradley and
Kaslow (22). Plasma levels of luteinizing hormone (LH), folliclestimulating hormone (FSH), and testosterone were determinedby using radioimmunoassay kits (Amersham Life Science andNippon DPC).

Adrenocorticotropic Hormone (ACTH) Stimulation Tests. Blood wascollected before and 1 h after the intraperitoneal injection of 25
␮g of ␤1–24 ACTH (Cortrosyn, Organon). Corticosterone wasmeasured by a radioimmunoassay (ICN Biomedicals).
Histology. Tissues were fixed with neutral-buffered formalin andembedded in paraffin. Sections were stained with hematoxylin
and eosin. Image capture and analysis were performed with
Adobe PhotoShop 3 image analysis software. The contour ofeach adipocyte was traced by hand and the cytoplasmic area wasdetermined.

Number and Motility of Sperm. The number and motility of
epididymal sperm were examined as described (23).
Tissue DNA and Lipids. Tissues were digested in SNET buffer (1%SDS兾400 mM NaCl兾5 mM EDTA兾20 mM Tris䡠HCl, pH 8.0)
containing proteinase K (0.2 mg兾ml). After extraction with788 兩 www.pnas.org

phenol兾chloroform, DNA concentrations were fluorimetricallydetermined with bisbenzimide H33258, using ultrapure calf
thymus DNA (Sigma) as the standard. Tissue lipids were extracted from approximately 100 mg of tissues according to themethod of Folch et al. (24) and then enzymatically measured.
Oxygen Consumption (VO2). VO2 and respiratory quotients (RQ)

were determined as previously described (25). Mice were fasted
or fed for 24 h. BRL35135A, a ␤3 agonist, was given intraperitoneally to the mice at 1 ␮g兾g of body weight.

Cold-Sensitivity Test. Mice were placed in a chamber whose

temperature was set at 4°C, and their core body temperature wasmonitored with a model 43 TA telethermometer and a model
402 colonic probe (Yellow Springs Instruments, Yellow Springs,
OH) every 1 h (26).

In Vitro Lipolysis. Adipocytes were isolated from epididymal fat
pads of male mice (12–16 weeks old) by using collagenase
digestion according to Rodbell’s method (27). One hundred
microliters of 10% (vol兾vol) cell suspension was incubated in a
final volume of 500 ␮l of a buffer containing 120 mM NaCl, 4mM KH2PO4, 1 mM CaCl2, 10 mM NaHCO3, 30 mM Hepes (pH7.4), 3% (wt兾vol), BSA and 1 unit兾ml adenosine deaminase, withor without 100 ␮M isoproterenol, at 37°C. Glycerol and FFA
were measured over a 15-min period.
In Vivo Lipolysis. After a 12-h fast, isoproterenol (0.3 mg兾kg) orsaline was injected intraperitoneally into control and HSL⫺兾⫺
mice 12–16 weeks old; 15 min later, blood was quickly collectedfrom the retroorbital venous plexus without anesthesia essentially as described by Suslic et al. (6). Plasma was separated forthe determination of glycerol and FFA.

Results
The gene encoding HSL was disrupted in embryonic stem cellsby homologous recombination. A portion of exon 5, and theentire exon 6, which encodes the catalytic domain (3, 11, 28, 29),
were replaced with a neo cassette (Fig. 1a). The intercross of theprogeny (HSL⫹兾⫺) resulted in offspring of both sexes with all
three genotypes at the HSL locus with the expected Mendelian
ratios (57:103:51; ␹2 ⫽ 0.23, P ⬎ 0.05) (Fig. 1b). Northern blot
analysis revealed that epididymal fat from HSL⫺兾⫺ mice lack awild-type transcript with a size of ⬇3.3 kb (data not shown).
Western blot analysis showed no immunoreactive HSL protein ineither epididymal fat (84 kDa) or testes (130 kDa) from
HSL⫺兾⫺ mice (Fig. 1c). NCEH activities were undetectable invarious organs, including WAT, BAT, and testes from the
HSL⫺兾⫺ mice, but were not reduced in macrophages in culture(Fig. 1d).
When HSL⫺兾⫺ male mice were mated with female mice, they
copulated normally, as evidenced by the formation of vaginalplugs, but none of the female mice mated to HSL⫺兾⫺ males
became pregnant. HSL⫺兾⫺ mice had smaller testes than
HSL⫹兾⫹ mice (Table 1). Sperm counts, in epididymis preparations, revealed that HSL⫺兾⫺ mice contained only 94 ⫾ 30
spermatozoa per epididymis, whereas HSL⫹兾⫹ mice contained
7.7 ⫾ 0.4 ⫻ 106 spermatozoa per epididymis. The sperm from the
HSL⫺兾⫺ epididymis were not motile, whereas 60% of thosefrom wild-type mice were motile. The thickness of the epithelial
layers of HSL⫺兾⫺ seminiferous tubules was greatly reduced
from 12 to 5–7 layers (Fig. 2). The number of mature spermatids
was also markedly reduced. Extensive vacuolation was observedin the epithelial cells. The vacuoles may contain a cholesterolester, since testicular cholesterol ester content was increased by
2.3-fold in the HSL⫺兾⫺ mice but the triacylglycerol content was
not (Table 1). This observation is consistent with the finding that
there was no difference in triacylglycerol lipase activities beOsuga et al.

MEDICAL SCIENCES

Fig. 1. Targeted disruption of the HSL gene. (a) Map of the HSL gene and targeting construct. The targeting vector was designed to delete HSL genomic
sequences including a part of exon 5 and the entire exon 6 encoding the catalytic motif (GXSXG). Targeted events generate an enzymatically inactive truncatedprotein lacking the C-terminal part of HSL. Tall boxes represent exons. The probe used for the diagnostic Southern blot is indicated as a shaded box. Restriction
sites: X, XbaI; E, EcoRI. (b) Southern blot of EcoRI-digested DNA. The wild-type genomic fragment is 5.9 kb, whereas the mutated gene is 2.6 kb. Genotypes areindicated below each lane. (c) Immunoblot analysis of white adipose tissue (WAT) and testes, showing the absence of immunoreactive HSL in ⫺兾⫺ mice. Notethat the testicular isoform (HSLtes) is larger than the adipocyte isoform (HSLadi) (130 kDa vs. 84 kDa). Molecular masses of the standards are indicated on the leftof the gel (5–15% acrylamide). (d) NCEH activities of WAT, brown adipose tissue (BAT) and testes were abolished in HSL⫺兾⫺ (open bars), whereas those of mouse
peritoneal macrophages (MPM) elicited by thioglycolate were comparable to wild-type (solid bars). Enzyme activity is expressed as picomoles of substratehydrolyzed per milligram of protein per minute. Data are represented as mean ⫾ SE. Numbers of mice used are as follows: 4 ⫹兾⫹ and 4 ⫺兾⫺ for WAT; 6 ⫹兾⫹and 5 ⫺兾⫺ for BAT; 3 ⫹兾⫹ and 3 ⫺兾⫺ for testes; and 7 ⫹兾⫹ and 8 ⫺兾⫺ for MPM.

tween wild-type and HSL⫺兾⫺ testes (38.6 ⫾ 4.5 vs. 32.8 ⫾ 6.5
nmol兾h per mg; P ⬎ 0.05, n ⫽ 5). HSL⫺兾⫺ epididymis contains
only degenerated spermatocytes and spermatids with a noticeable lack of mature sperms (Fig. 2). There were no detectable
abnormalities in the interstitial cells, including the Leydig cells,in HSL⫺兾⫺ mice. Plasma levels of testosterone, folliclestimulating hormone, and luteinizing hormone were not significantly different between the wild-type and HSL⫺兾⫺ mice,indicating that the oligospermia did not result from a hormonal
insufficiency. Furthermore, plasma corticosterone levels were
not different between the wild-type and HSL⫺兾⫺ mice before(143 ⫾ 31 vs. 109 ⫾ 18 ng兾ml; P ⬎ 0.05, n ⫽ 8) or after
stimulation with adrenocorticotropic hormone (460 ⫾ 61 vs.
387 ⫾ 17 ng兾ml; P ⬎ 0.05, n ⫽ 8).
Because HSL is highly expressed in adipose tissues, we examined the weight and morphology of BAT and WAT. BAT wasTable 1. Triacylglycerol, free cholesterol, and cholesterol ester contents in testis, BAT, and WAT
Tissue lipids, mg兾g tissue
TissueGenotypen

Weight, mg

TG

FC

EC

Testis

⫹兾⫹
⫺兾⫺
⫹兾⫹
⫺兾⫺
⫹兾⫹
⫺兾⫺

5
5
4
4
4
4

128 ⫾ 686 ⫾ 4*103 ⫾ 15170 ⫾ 18**356 ⫾ 36277 ⫾ 45

4.9 ⫾ 1.6
4.2 ⫾ 0.9457 ⫾ 15548 ⫾ 27**512 ⫾ 137
798 ⫾ 75

9.4 ⫾ 0.2
10.2 ⫾ 0.2
0.13 ⫾ 0.02
0.04 ⫾ 0.02**
0.07 ⫾ 0.04
0.19 ⫾ 0.09

3.0 ⫾ 0.4
6.9 ⫾ 1.2**
0.08 ⫾ 0.01
0.07 ⫾ 0.01
0.04 ⫾ 0.03
0.12 ⫾ 0.06

BAT
WAT

Tissue levels of total cholesterol (TC), free cholesterol (FC), and triacylglycerol (TG) were measured enzymatically. Esterified cholesterol (EC) levels were
calculated from TC and FC values. Values are expressed as mean ⫾ SE. *, P ⬍ 0.001; **, P ⬍ 0.05 versus HSL⫹兾⫹ by Student’s t test.
Osuga et al.

PNAS 兩 January 18, 2000 兩 vol. 97 兩 no. 2 兩 789

Fig. 2. Histology of epididymis (⫻50) and testis (⫻100). HSL⫺兾⫺ epididymis
contains no sperms but degenerated spermatocytes and spermatids. The
thickness of the epithelial layers of the HSL⫺兾⫺ seminiferous tubules was
greatly reduced from 12 to 5–7 layers. The number of mature spermatids was
also markedly reduced. Extensive vacuolation was observed in the epithelial
cells.

significantly enlarged in HSL⫺兾⫺ mice by 1.65-fold, primarily
because of cellular hypertrophy, because triacylglycerol content
was increased (Table 1) but DNA content was not (Table 2). In
support of this observation, the median cytoplasmic area of BAT
adipocytes was increased by about 5-fold in HSL⫺兾⫺ mice (Fig.
3a). Less remarkable cellular hypertrophy was observed in
HSL⫺兾⫺ WAT, where the median cytoplasmic area was enlarged by 2-fold (Fig. 3b). Nevertheless, the WAT mass was notincreased in the gonadal, retroperitoneal, or femoral regions(Table 2).
Body weight was not significantly different, at least until 24
weeks of age, between wild-type and HSL⫺兾⫺ mice (36.1 ⫾ 2.2
vs. 35.7 ⫾ 1.2 g, P ⬎ 0.05, n ⫽ 13 in males; 29.3 ⫾ 1.0 vs. 26.7 ⫾0.8 g, P ⬎ 0.05, n ⫽ 14 in females). Overall VO2 (1.09 ⫾ 0.05 vs.
1.23 ⫾ 0.07 ml兾min per mouse; P ⬎ 0.05, n ⫽ 6) and RQ (1.20 ⫾0.02 vs. 1.12 ⫾ 0.03; P ⬎ 0.05, n ⫽ 6) were not different between
the wild-type and HSL⫺兾⫺ mice fed a normal chow. They were
not different even when fasted or treated with BRL35135A, a␤3-agonist (data not shown). Core body temperature changed
from 38.5 ⫾ 0.2°C to 38.1 ⫾ 0.2°C in the wild-type mice (n ⫽ 6),
and it changed from 38.3 ⫾ 0.1°C to 37.6 ⫾ 0.3°C in the HSL⫺兾⫺
mice (n ⫽ 6) after exposure to an atmospheric temperature of
4°C. Thus, the HSL⫺兾⫺ mice were as cold-sensitive as HSL⫹兾⫹mice.
To determine how much triacylglycerol hydrolysis is accounted for by HSL in adipocytes, we measured triacylglycerol
lipase activities in the presence of 1 M NaCl, which selectively
inhibits LPL, and compared the lipolysis both in vitro and in vivo
(Fig. 4). The HSL⫺兾⫺ WAT expressed significantly lower

Fig. 3. Enlarged lipid droplets in adipocytes from BAT (a) and epididymal fat(WAT) (b) in wild-type and HSL⫺兾⫺ mice. (⫻50.) Distribution of cytoplasmic
area is shown in the graphs: F, wild-type; E, HSL⫺兾⫺. Median cytoplasmic area
was increased by about 5-fold in BAT and by 2-fold in WAT.

(40%), but still substantial, activities of triacylglycerol lipase ascompared with the wild-type WAT (Fig. 4a). In BAT, there wasno significant difference in the triacylglycerol lipase activities
between wild-type and HSL⫺兾⫺ mice. In support of theseresults, adipocytes isolated from HSL⫺兾⫺ WAT released reduced, but still substantial, amounts of glycerol as well as FFA
into the incubation media in vitro (Fig. 4b). Release of FFA, but
not of glycerol, was stimulated by treatment with isoproterenolin the HSL⫺兾⫺ adipocytes. The isoproterenol-stimulated increment in the FFA concentration in HSL⫺兾⫺ WAT was
smaller than that in wild-type WAT. In vivo, HSL⫺兾⫺ mice had
lower plasma glycerol and FFA levels than wild-type mice (Fig.
4c). Although the increase in plasma glycerol concentration after
the injection of isoproterenol was very small in HSL⫺兾⫺ miceas compared with wild-type mice, the increase in plasma FFA
concentration was at least as great as that in the wild-type mice.
Discussion
Extremely divergent functions have been assigned to HSL: (i)liberation of FFA by triacylglycerol lipase activity in adipocytes,(ii) breakdown of cholesterol ester to supply cholesterol forsteroidogenesis in adrenals, ovaries, and possibly testes, and (iii)
breakdown of cholesterol ester in cells such as macrophages toprevent foam cell formation in arterial walls. Furthermore, it ishighly expressed in spermatids and spermatozoa as a larger
testicular isoform (HSLtes). In the current study, we have gen-Table 2. Tissue mass and DNA content of adipose tissues in various sites
TissueGenotypen

Fat mass, mg

Tissue DNA, ␮g

Mass兾DNA ⫻ 103

BAT

⫹兾⫹
⫺兾⫺
⫹兾⫹
⫺兾⫺
⫹兾⫹
⫺兾⫺
⫹兾⫹
⫺兾⫺

4
4
4
4
4
4
4
4

72 ⫾ 3139 ⫾ 15*371 ⫾ 85180 ⫾ 1384 ⫾ 2871 ⫾ 8256 ⫾ 47362 ⫾ 69

183 ⫾ 2196 ⫾ 20147 ⫾ 17126 ⫾ 1141 ⫾ 444 ⫾ 2358 ⫾ 38382 ⫾ 32

0.39 ⫾ 0.02
0.71 ⫾ 0.05*
2.44 ⫾ 0.34
1.47 ⫾ 0.15**
1.92 ⫾ 0.52
1.61 ⫾ 0.11
0.70 ⫾ 0.06
1.00 ⫾ 0.26

Epididymal WAT
Retroperitoneal WAT
Femoral WAT

Tissue DNA content was measured fluorimetrically. Values are expressed as mean ⫾ SE. *, P ⬍ 0.001; **, P ⬍ 0.05 versus HSL⫹兾⫹ by Student’s t test.
790 兩 www.pnas.org

Osuga et al.

erated mice lacking HSL and used these animals to test thesevarious functions.
The most striking phenotype of HSL⫺兾⫺ mice is male sterility
caused by oligospermia. Holst et al. (10, 11) have shown that HSL
is localized in elongating spermatids and spermatozoa, but not
in interstitial cells such as Leydig cells. Consistent with theabsence of HSL from Leydig cells, HSL⫺兾⫺ mice have normal
plasma levels of testosterone, follicle-stimulating hormone, andluteinizing hormone. Thus the oligospermia does not result fromhypogonadism. Rather, it is conceivable that HSL is directly
involved in spermatogenesis in a manner similar to that of othergenes whose functions are crucial for male germ cell differentiation but are not needed for oogenesis (30). Because HSL
catalyzes the hydrolysis of cholesterol ester and triacylglycerol, it
is reasonable to speculate that cholesterol or fatty acids releasedby the action of HSL are required for spermatogenesis. In thisconcern, it is noteworthy that cholesterol contents of sperms are
significantly altered during epididymal maturation (31) and that
deficiency of essential fatty acids leads to extensive testiculardegeneration (32). It is also possible that the male sterility arises
from a disturbance of retinoid metabolism, since HSL mediates
retinol ester hydrolysis (33). It is well known that vitamin A isessential for reproductive function. For example, vitamin A-deficient rats develop a wide variety of phenotypes including malesterility (34). In addition, male sterility is the major phenotypeof retinoid receptor RAR␣ (35) and RXR␤ (36) knockout micethat survive perinatal lethality. Further studies are needed toclarify the mechanism by which HSL deficiency causes malesterility.
Durham and Grogan (37) reported that two types of cholesterol ester hydrolases are present in the testes: temperaturestable and temperature-labile (TLCEH). TLCEH seems to be
distinct from HSL, because of its different biochemical properOsuga et al.

ties (38) and its localization in Sertoli cells (37). However, HSL
may be related to TLCEH, because both enzymes have thefollowing characteristics in common. They are more active at low
temperature (28, 37) and their activity increases with sexual
maturity (8, 39). When measured at 37°C, NCEH activities werebarely detectable in the testes of HSL⫺兾⫺ mice (Fig. 1d), further
supporting the hypothesis that HSL is related to TLCEH.
Phenotypes in adipocytes, where HSL is highly expressed,
were milder than expected. Even though NCEH activities werecompletely absent and the mean size of adipocytes was increased, the adipose tissue mass was increased only in BAT and
not in WAT in HSL⫺兾⫺ mice. In agreement with these observations, HSL⫺兾⫺ mice were not obese, judged from the growth
curves. Since uncoupling protein (UCP) carries out thermogenesis using fatty acids as substrates in BAT, HSL⫺兾⫺ mice were
expected to be sensitive to cold. However, this was not the case.
Furthermore, there was no difference in the overall VO2, RQ,
and core body temperature between the wild-type and HSL⫺兾⫺
mice, irrespective of the feeding status. These results indicatethat fatty acids supplied from HSL-mediated lipolysis are not the
only substrates utilized for the thermogenesis in BAT. Similar
enlargement of lipid vacuoles in BAT adipocytes has beenreported for mice lacking norepinephine and epinephrine (40)and UCP1 knockout mice (26). However, these mice are reportedly cold sensitive. Therefore, adipocyte hypertrophy in BAT
and cold sensitivity appear to be independent phenotypes.
Phenotypes in WAT are interesting. Although the mean sizeof adipocytes in WAT was increased, total tissue mass was not.
These results suggest the heterogeneity of adipocytes in WAT;a certain cell population is sensitive to lipid accumulation andthe other is not. Alternatively, triacylglycerol may accumulate
beyond the limit that the adipocytes can tolerate, thereby leading
PNAS 兩 January 18, 2000 兩 vol. 97 兩 no. 2 兩 791

MEDICAL SCIENCES

Fig. 4. The redundancy of triacylglycerol lipase. (a) Triacylglycerol lipase activities in WAT and BAT from the wild-type (solid bar) and HSL⫺兾⫺ mice (open bar)(n ⫽ 5). (b) In vitro lipolysis in adipocytes isolated from epididymal fat pad of wild-type (⫹兾⫹; n ⫽ 12) and HSL⫺兾⫺ mice (⫺兾⫺; n ⫽ 8). Adipocyte suspensions
were treated with 100 ␮M isoproterenol (solid bar; ISO) or without it (open bar; none) for 15 min. Glycerol and FFA concentrations in the media were determined.
(c) In vivo lipolysis. After a 12-h fast, 0.3 mg兾kg of isoproterenol (solid bar; ISO) or saline (open bar; Saline) was injected intraperitoneally into control (⫹兾⫹; n ⫽7) and HSL⫺兾⫺ (⫺兾⫺; n ⫽ 10) mice. After 15 min, blood was collected and plasma concentrations of glycerol and FFA were determined. Data are expressed asmean ⫾ SE. Mice used in these experiments were 12–16 weeks old. *, P ⬍ 0.001; **, P ⬍ 0.05 by Student’s t test.

to disruption of cell integrity—i.e., death—in a population of
adipocytes of HSL⫺兾⫺ mice.
It is noteworthy that WAT from HSL⫺兾⫺ mice retained
substantial activities that hydrolyzed triacylglycerol in the presence of 1 M NaCl, an inhibitor of LPL (Fig. 4a). In agreementwith these results, adipocytes isolated from HSL⫺兾⫺ epididymal fat released significant amounts of glycerol and FFA into the
incubation media (Fig. 4b). This residual lipase activity appearedslightly responsive to isoproterenol. This is also consistent with
the in vivo lipolysis experiments demonstrating that plasma FFA
and glycerol were increased in response to isoproterenol treatment (Fig. 4c). Together, these results strongly indicate that HSL
is not the only molecule that hydrolyzes triacylglycerol in adipocytes. This may be one of the reasons why HSL⫺兾⫺ mice wereneither obese nor cold sensitive.
BAT expressed significantly lower activities of NCEH than
WAT, and these activities were completely eliminated by the
HSL disruption (Fig. 1d). On the other hand, BAT contained
higher activities of triacylglycerol lipase than did WAT, but these
activities were not affected by the HSL disruption (Fig. 4a).
Considering these observations together, we speculate that thehydrolysis of triacylglycerol in BAT is primarily mediated by an
enzyme that is distinct from HSL. This is also very consistent
with the lack of cold sensitivity in HSL⫺兾⫺ mice.
Cholesterol-ester-laden foam cells are a hallmark of atherosclerosis. Cholesterol is in a dynamic equilibrium between freeand esterified forms. The esterification and hydrolysis are mediated by acyl-CoA:cholesterol acyltransferase (ACAT) and
NCEH, respectively. Therefore, it is believed that NCEH plays
an important role in atherogenesis. Interestingly, NCEH activities expressed in arterial wall as well as in macrophage-like celllines are hormone sensitive and are activated by protein kinase
A (13, 14, 41, 42). Furthermore, Small et al. (16) have shown that
the NCEH activities are completely inhibited by an anti-HSL
antibody in the mouse macrophage cell line WEHI. Together
with the fact that HSL is expressed in mouse peritoneal macrophages and P388D1 and J774 macrophage cell lines (17), it has
been widely accepted that HSL mediates NCEH activity inmacrophages. In contrast to this belief, our current results
strongly indicate that the NCEH activity is accounted for by amolecule(s) distinct from HSL at least in mouse peritoneal
macrophages (Fig. 1d). In this respect, it is noteworthy thathuman monocyte-derived macrophages may (43) or may not (19)express HSL, and their NCEH activity is not hormone sensitive (19).
In conclusion, we demonstrate that HSL is essential for
spermatogenesis but is dispensable for lipolysis in adipocytes.
These results also help to shed light on the debate about whether
NCEH activity in macrophages is HSL.

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792 兩 www.pnas.org

We thank Y. Tsutsumi for determination of RQ, and K. Saito, M.
Kusubae, and E. Herai for maintenance of animals. We also thank T.
Kitamine, T. Gotoda, Y. Iizuka, Z. Chen, K. Ohashi, K. Harada, S.
Perrey, Y. Tamura, and H. Shimano for helpful discussions. This work
was supported by a Grant-in-Aid for Scientific Research from the
Ministry of Education, Science and Culture, by the Promotion of
Fundamental Studies in Health Science of The Organization for Pharmaceutical Safety and Research, by Health Sciences Research Grants(Research on Human Genome and Gene Therapy) from the Ministry of
Health and Welfare, by the Research Service of the Department of
Veterans Affairs, and by Grants DK 46942 and 49705 from the National
Institutes of Health.

Osuga et al.