Hormone-sensitive lipase is involved in hepatic cholesterylester hydrolysis
Motohiro Sekiya,* Jun-ichi Osuga,* Naoya Yahagi,* Hiroaki Okazaki,* Yoshiaki Tamura,*
Masaki Igarashi,* Satoru Takase,* Kenji Harada,* Sachiko Okazaki,* Yoko Iizuka,* Ken Ohashi,*
Hiroaki Yagyu,† Mitsuyo Okazaki,§ Takanari Gotoda,* Ryozo Nagai,** Takashi Kadowaki,*Hitoshi Shimano,†† Nobuhiro Yamada,†† and Shun Ishibashi1,*,†

Abstract Hormone-sensitive lipase (HSL) regulates thehydrolysis of acylglycerol and cholesteryl ester (CE) in various organs, including adipose tissues. However, the hepatic
expression level of HSL has been reported to be almost
negligible. In the present study, we found that mice lacking both leptin and HSL (Lepob/ob/HSL2/2) showed massive accumulation of CE in the liver compared with Lepob/ob/
HSL1/1 mice, while triacylglycerol (TG) accumulation wasmodest. Similarly, feeding with a high-cholesterol diet induced hepatic CE accumulation in HSL2/2 mice. Supportingthese observations, we detected significant expression of protein as well as mRNA of HSL in the liver. HSL2/2 mice
showed reduced activity of CE hydrolase, but not of TG lipase, in the liver compared with wild-type mice. Furthermore,
we confirmed the expression of HSL in viable parenchymal
cells isolated from wild-type mice. The hepatocytes from
HSL2/2 mice showed reduced activity of CE hydrolase andcontained more CE than those from HSL1/1 mice even without
the incubation with lipoproteins. Incubation with LDL further
augmented the accumulation of CE in the HSL-deficient
hepatocytes. From these results, we conclude that HSL is involved in the hydrolysis of CE in hepatocyes.—Sekiya, M., J-i.
Osuga, N. Yahagi, H. Okazaki, Y. Tamura, M. Igarashi, S.
Takase, K. Harada, S. Okazaki, Y. Iizuka, K. Ohashi, H. Yagyu,
M. Okazaki, T. Gotoda, R. Nagai, T. Kadowaki, H. Shimano, N.
Yamada, and S. Ishibashi. Hormone-sensitive lipase is involved
in hepatic cholesteryl ester hydrolysis. J. Lipid Res. 2008. 49:
1829–1838.
Supplementary key words
ob/ob • hepatocytes

fatty liver • leptin • mouse • lipoproteins •This work was partially supported by Grants-in-Aid from the Japan Health
Science Foundation and by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists (to M.S.). This work was also supported
by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture and by the Program for the Promotion of Fundamental Studies
in Health Sciences of the National Institute of Biomedical Innovation.
Manuscript received 22 April 2008 and in revised form 9 May 2008.
Published, JLR Papers in Press, May 9, 2008.
DOI 10.1194/jlr.M800198-JLR200

The mammalian liver plays a central role in maintaining
the whole body cholesterol homeostasis. The liver delivers
cholesterol to peripheral tissues via VLDL particles, andthe excess peripheral cholesterol is transported to the liver
through HDL acceptors (1). Cholesterol balance in theliver involves three major processes: 1) the input pathway,
which consists of de novo synthesis and lipoprotein uptake
from the periphery; 2) the output pathway, which consistsof secretion into the circulation as VLDL and into the bile;
3) reversible conversion of cholesterol to cholesteryl esters(CEs). Esterification of cholesterol with long-chain fattyacyl-CoA is accomplished by ACAT (2). Newly formed
CEs are secreted as a component of VLDL or are stored
in intracellular lipid droplets in the cytoplasm, whereCEs undergo a constant cycle of hydrolysis and resynthesis.
To date, at least four major CE hydrolases have been
identified in the liver and shown to differ in their subcellular localizations, functions, and enzymatic properties.
1) The lysosomal acid lipase (LAL) is involved in the hydrolysis of CEs and triacylglycerols (TGs) delivered tothe hepatocytes via receptor-mediated endocytosis of lipoproteins (3). 2) The cytosolic CE hydrolase from rat liver
has also been purified and characterized. This lipase has
been considered to be a key enzyme for releasing free cholesterol from the stores of CEs in the cytoplasm (4). 3) The
microsomal CE hydrolase has also been purified fromrat liver and characterized (5). 4) Carboxyl ester lipase(CEL) is another cholesterol esterase present in the he-

Abbreviations: ALT, alanine aminotransferase; ATGL, adipose triglyceride lipase; CE, cholesteryl ester; CEL, carboxyl ester lipase; DG,
diacylglycerol; HSL, hormone-sensitive lipase; LAL, lysosomal acid lipase; LPDS, lipoprotein-deficient serum; NPC, nonparenchymal cell;PC, parenchymal cell; SREBP, sterol-regulatory element binding protein; TG, triacylglycerol.
1
To whom correspondence should be addressed.
e-mail: ishibash@jichi.ac.jp

Copyright © 2008 by the American Society for Biochemistry and Molecular Biology, Inc.
This article is available online at http://www.jlr.org

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Departments of Metabolic Diseases,* and Cardiovascular Medicine,** Faculty of Medicine, University ofTokyo, Tokyo 113-8655, Japan; Laboratory of Chemistry,§ Department of General Education, Tokyo Medical
and Dental University, Chiba 272-0827, Japan; Department of Metabolism, Endocrinology, and Atherosclerosis,††Institute of Clinical Medicine, University of Tsukuba, Ibaragi 305-8575, Japan; and Division of Endocrinology
and Metabolism,† Department of Medicine, Jichi Medical University, Tochigi 329-0498, Japan(16). Mice were housed in a temperature-controlled environment
with a 12 h light/dark cycle and allowed free access to water anda standard chow diet (Oriental MF, composed of 60% carbohydrate, 13% fat, and 27% protein on a caloric basis; Oriental Yeast,
Tokyo, Japan). Mice were euthanized at 16 weeks after a 6 h fast,
except in the fasting/refeeding study, in which mice were starved
for 24 h or refed for 12 h after a 24 h starvation.

Dietary cholesterol load experiments
Eight-week-old female HSL2/2 mice and their littermate wildtype mice were fed a standard chow with or without 2% cholesterol for 7 weeks. Mice were euthanized after a 12 h starvation.
All experiments were performed in accordance with our institutional guidelines.

Liver lipid content
Liver lipid was extracted by the chloroform-methanol method,
and total cholesterol and free cholesterol were determined byfluorometric microassay according to a modified method ofHeider and Boyett (17), with some modifications as described
previously (18).

Immunoblotting of HSL
Livers (two mice for each genotype) were excised from 16 week
old male mice after 6 h of fasting, homogenized in buffer A(20 mM Tris-HCl, pH 7.0, 250 mM sucrose, 5 mg/ml leupeptin,
and 2.8 mg/ml aprotinin), and microcentrifuged at 14,0003g for
15 min. Fifty micrograms of the supernatant fraction was subjected to SDS-PAGE analysis. Homogenate (10 mg) from epididymal white adipose tissues of a wild-type male mouse (16 weeksold) was used as a control. Immunoblot analysis was performed
using an ECL kit (Amersham Biosciences, Piscataway, NJ) (13).

Hepatocyte isolation
Isolation of viable PCs and NPCs from mouse liver was performed as described previously (19, 20). In brief, the portal vein
was cannulated, and the liver was perfused with 0.05% collagenase (Wako Pure Chemicals, Tokyo, Japan). After removing undigested tissues, the cell suspension was centrifuged at 1003g for
2 min. The pellet was used for PC preparation, while for NPC
preparation the supernatant was filtered through a nylon meshto eliminate PCs. The cell suspension was layered on top of
70% (v/v) Percoll (Amersham Bioscience, Tokyo, Japan) andthen centrifuged at 8003g for 10 min. The pellet was saved as
intact PCs. The supernatant located on the top of Percoll,
which contained most of the NPCs but also some PCs and debris, was further centrifuged through a two-step Percoll gradient(25% 1 50%) at 8003g for 30 min. A pure NPC band at the interface between the two density cushions was collected. The cells
were washed twice with PBS and pelleted by centrifugation. The
purity of the cells was morphologically assessed by light microscopic examination. More than 90% of the cells in the PC fraction were hepatocytes, whereas less than 10% of the cells in the
NPC fraction were hepatocytes. The cell pellet was redissolvedin buffer A and sonicated with a Branson sonifier at power
2 for two 10 s pulses. After microcentrifugation at 14,0003g for
10 min, an aliquot of the supernatant was analyzed by immunoblot analysis.

EXPERIMENTAL PROCEDURES
Northern blot analysis
Animals
Cross-breeding experiments used leptin-deficient Lepob/ob mice.
HSL2/2 mice (13) and Lepob/ob/HSL2/2 mice (16) were generated, and genotyping was performed as described previously

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Total RNA was isolated using Trizol Reagent (Life Technologies, Helgerman, CT). Ten microgram RNA samples equallypooled from four to six mice of each genotype were electrophoresed on a 1% agarose gel containing formaldehyde andDownloaded from www.jlr.org at Univ of Tokyo Medical Library on October 1, 2008

patic extralysosomal membrane compartment and possesses the capacity to hydrolyze HDL-CE (6).
Hormone-sensitive lipase (HSL) is an intracellular neutral lipase that catalyzes the hydrolysis of cellular CEs aswell as TG, diacylglycerol (DG), monoacylglycerols, andother lipids. HSL is diffusely distributed throughout thecytosol, and upon activation, translocation of HSL fromthe cytosol to lipid droplets occurs (7, 8). Although HSL
was initially identified as an adipose-specific lipase, it has
been clarified that HSL is also expressed and functions
in a wide variety of organs and cells, including heart,skeletal muscle, adrenal glands, testes, ovaries, pancreaticb-cells, and intestines (8–10). Despite the broad tissue distribution, the liver has been considered to lack HSL (11),although observations suggesting the importance of HSL
in liver were reported (12).
Several laboratories, including ours, have generated HSLdeficient (HSL2/2) mice by targeted gene disruption (12–15). Despite the decreased ability of their adipocytes torelease FFAs, these mice were not obese. We have further generated leptin/HSL double mutant mice (Lepob/ob/
HSL2/2) by cross-breeding leptin-deficient Lepob/ob micewith HSL2/2 mice (16) to elucidate the role of HSL, especially in the setting of obesity. Unexpectedly, the Lepob/ob/
HSL2/2 mice gained less adiposity than Lepob/ob mice, withmassive accumulation of preadipocytes in adipose tissue,and HSL deficiency also suppressed the feeding activityof Lepob/ob mice.
Here, we show that HSL plays a pivotal role as a lipase in
the liver, especially as a CE hydrolase. Massive accumulation of CEs was observed in the liver of Lepob/ob/HSL2/2
mice, while the hepatic TG content of Lepob/ob/HSL2/2
mice was similar to that of Lepob/ob/HSL1/1 mice. Moreover, feeding diets containing high cholesterol resulted
in the accumulation of hepatic CEs in HSL2/2 mice. In
accordance with these results, CE hydrolase activity wasdecreased by HSL deficiency, while TG lipase activity was
unchanged. These results suggested that HSL could serve
as a CE hydrolase in hepatocytes. As expected, the expression of HSL is detected in liver homogenates, andparenchymal cells (PCs) contain more HSL protein than
nonparenchymal cells (NPCs). Furthermore, to eliminate
indirect effects from other tissues, we isolated primary
hepatocytes. The CE hydrolase activity is reduced in thehepatocytes from HSL2/2 mice. Intracellular CE content
is increased even in the absence of lipoproteins in thehepatocytes from HSL2/2 mice, and upon exposure to
LDL, a more pronounced increase was observed. Basedon these findings, we conclude that HSL is definitely
expressed in hepatocytes and involved in CE hydrolysis
in the liver.

transferred to a nylon membrane. The membranes were hybridized with probes that were labeled with [a-32P]dCTP using the
Megaprime DNA Labeling System kit (Amersham Biosciences)in Rapid-hyb Buffer (Amersham Biosciences) and analyzed usinga BAS2000 Bioimaging Analyzer (Fuji Photo Film, Tokyo, Japan).
Loading was normalized by the expression of 36B4 (acidic ribosomal phosphoprotein P0) mRNA.

TG lipase and neutral CE hydrolase activity assay

Statistics
Microsomal ACAT activity
Liver microsomal protein was obtained by ultracentrifugation
at 100,000 g for 45 min. The precipitates were resuspended andultracentrifuged again at 100,0003g for 45 min. The precipitates
were resuspended and used for the assay. ACAT activity in microsomes was determined by the rate of incorporation of [1-14C]
oleoyl-CoA into the CE fraction according to Yagyu et al. (18).

Biochemical analyses
Blood was collected from the retro-orbital venous plexus
after a 6 h or a 12 h fast. Cholesterol (Determiner TC; Kyowa
Medex, Tokyo, Japan), TG (TG LH; Wako Pure Chemicals),
FFA (NEFA C; Wako Pure Chemicals), and alanine aminotransferase (ALT) (transaminase C-II: Pyruvate oxidase Pediococcus sp.
(POP) N-(2-hydroxy-3-sulfopropyl)-m-toluidine (TOOS) method;
Wako Pure Chemicals) were measured enzymatically. HPLC was
performed as described previously (21).

TLC
Liver lipid was extracted by the chloroform-methanol method,
and aliquots were pooled for each genotype. Evaporated lipid
was dissolved in chloroform, and lipid from 0.6 mg of liver tissue
was loaded in each lane. Lipids were separated by TLC withhexane-diethylether-acetic acid (80:20:1) or chloroform-acetoneacetic acid (96:4:1) as solvent. Visualization was done with 10%
sulfuric acid.

Histology
Mice (16 weeks) were euthanized after a 6 h fast, and their livers
were excised and fixed in 10% neutral buffered formalin and embedded in paraffin. Sections were stained with hematoxylin-eosin.

Primary culture of hepatocytes and CE formation
Hepatocytes were isolated from nonfasted HSL2/2 mice andtheir littermate HSL1/1 mice (10 weeks) by the collagenase perfusion method as described previously (22). Cells were resuspended in Williamsʼ E medium supplemented with 5% fetal
calf serum, and seeded on collagen-coated dishes at a final density of 2 3 104 cells/cm2, and attached for 6 h.

Statistical differences between groups were analyzed by one-way
ANOVA and a posthoc Tukey-Kramer test, unless stated otherwise.

RESULTS
Both leptin deficiency and dietary cholesterol load induce
massive accumulation of CEs in the liver of HSL2/2 mice
Leptin-deficient Lepob/ob mice exhibit many characteristics of obesity, including hepatic steatosis (25). To determine the effect of HSL deficiency on hepatic lipid
metabolism, especially in a leptin-deficient obesity model,
HSL2/2 mice were cross-bred with leptin-deficient Lepob/ob
mice and the liver lipid content of each genotype wasmeasured (6 h fasted mice; Fig. 1A). To our surprise, inthe Lep ob/ob background, the hepatic CE content was
remarkably increased (173% for males and 214% forfemales) in Lepob/ob/HSL2/2 mice compared with Lepob/ob/
HSL1/1 mice. There was no difference in acylglycerol content between Lepob/ob/HSL1/1 and Lepob/ob/HSL2/2 mice. In
the Lep1/1 background, acylglycerol content was reducedin HSL2/2 mice compared with HSL2/2 mice, consistent withprevious reports (26, 27). There was a trend toward an increase in hepatic CE content in HSL2/2 compared with
HSL1/1 mice, especially in female mice (17% for males
and 137% for females). Free cholesterol content was not affected by either the absence or the presence of HSL or leptin.
HSL2/2 mice have reduced plasma FFA levels caused by
the impediment of the mobilization of FFA from adipose
tissues (12–16). In the fasted state, HSL normally stimulates
adipose lipolysis, thus increasing the influx of FFA to theliver and contributing to hepatic TG accumulation. Therefore, we evaluated both fasted and refed states (fasting, 24 hof fasting; refeeding, 12 h of feeding after 24 h of fasting;Fig. 1B). In the fasted state, hepatic TG and CE tended toaccumulate to a greater extent, and the differences between
Hormone-sensitive lipase in liver

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Enzyme activity was assayed basically as described previously(13) with some modifications in the sample preparation. In
brief, liver S-100 protein in buffer A was obtained by ultracentrifugation at 100,0003g for 45 min. Buffer exchange to PBS containing 1 mM EDTA was carried out by dialysis for 4 h, because
the liver homogenate turned out to include some unknown factor(s) that interfered with assay linearity (data not shown). The
samples were incubated at 37°C for 30–60 min in a final volume of 200 ml of a reaction mixture containing 105 mmol/ltri[3H]oleoylglycerol (99.4 mCi/mmol), 23.7 mmol/l lecithin,
12.5 mmol/l sodium taurocholate, 1 mol/l NaCl, and 85 mmol/l
potassium phosphate (pH 7.0). The high concentration of NaCl
was included to inactivate LPL. CE hydrolase activity was measured using a reaction mixture containing 6.14 mmol/l cholesterol [1-14C]oleate (48.8 mCi/mmol).

For the neutral CE hydrolase assay, the attached cells weremaintained in Medium 199 containing Earleʼs salts (Invitrogen)and 5% fetal calf serum. After incubation for 20 h, the medium
was replaced with the same medium and the cells were further
incubated for 12 h. The cells were washed twice with PBS,
scraped into buffer A, and sonicated with a Branson sonifier
at power 2 for two 10 s pulses. After microcentrifugation at
14,000 g for 10 min, an aliquot of the supernatant was analyzed
for neutral CE hydrolase assay.
For CE formation, attached cells were incubated in Williamsʼ
E medium containing 5% human lipoprotein-deficient serum(LPDS) for 48 h and subsequently incubated in the same medium containing human LDL (100 mg/ml) and [1-14C]oleatealbumin complex with vehicle or 10 mM CS-505, an ACAT
inhibitor, which were provided by Sankyo and Kyoto Pharmaceuticals (Tokyo, Japan), for 24 h as described previously (23)with minor modifications. Intracellular lipids were separated by
TLC, and spots corresponding to the CE fraction were scraped
and radioactivities were measured. LPDS (d . 1.21 g/ml) andLDL (d 5 1.019–1.063 g/ml) were isolated with sequential ultracentrifugation from plasma of healthy human volunteer subjects (24).
Informed consent was obtained from the volunteers according to
our institutional guideline for the use of human materials.

HSL1/1 and HSL2/2 mice were more pronounced than in
the refed state. In both states, the accumulation of CEs in
the HSL2/2 mice described above was conserved.
Moreover, hepatic accumulation of CEs in HSL2/2
mice was confirmed with the diet-induced model. HSL2/2
mice were challenged with dietary cholesterol (2%) for
7 weeks (euthanized after a 12 h fast; Fig. 1C). Dietarycholesterol induced hepatic CE accumulation in HSL2/2
mice (217% increase).
HSL is expressed in PCs
Liver has been reported to express HSL at a negligible
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patic HSL expression. Therefore, we performed immunoblot analysis of liver homogenates (Fig. 2A). The hepatic
expression of HSL was detected in both Lep1/1 andLepob/ob mice using our polyclonal anti-HSL antibody, andthe expression level was not affected by the presence orabsence of leptin. Quantification of the immunoblotting
signals indicated that the expression levels of HSL in liver
were approximately 5% of those in adipose tissue.
HSL has been considered to be present in macrophages(13, 28–30), although there are some conflicting reports(31, 32). Since Kupffer cells are the resident macrophages
in liver, we examined whether or not HSL was expressedin purified hepatocytes. After digesting liver by collage-

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Fig. 1. Liver lipid contents of each genotype in the cross-breeding experiment with Lepob/ob mice. A: After a
6 h fast (n 5 10, 16 weeks of age). B: Fasting/refeeding study (n 5 4, 16 weeks of age; female mice werestarved for 24 h or refed for 12 h after a 24 h starvation). C: Liver lipid content of hormone-sensitive lipasedeficient (HSL2/2) mice fed a high-cholesterol diet (n 5 5–6; female mice were fed a standard chow dietwith or without 2% cholesterol for 7 weeks.). Livers were excised, and lipid was extracted by the chloroformmethanol method. Total cholesterol and free cholesterol (FC) were determined by fluorometric microassay.
CE, cholesteryl ester; TG, triacylglycerol. Data are expressed as means 6 SEM. * P , 0.05, ** P , 0.01 as
determined by ANOVA followed by the Tukey-Kramer posthoc test.

nase perfusion, viable PCs and NPCs were purified on a
Percoll gradient. The expression of HSL was detected in
PCs as well as in NPCs, which contain Kupffer cells, andHSL was also expressed in H2.35 and McARH7777 murine
hepatocyte cell lines (Fig. 2B). HSL was more highly expressed in PCs than in NPCs (Fig. 2B). Taken together,
these findings indicate that HSL is indeed expressed inhepatocytes and contributes especially to hepatic CE hydrolase activity. For some samples from whole liver homogenates and liver PCs, the bands immunoreactive with ouranti-HSL antibody were duplets. For samples from NPCs,
McARH7777, H2.35, or mouse peritoneal macrophages,
however, the bands were single, with the same molecularweight as the upper bands of the duplets. We speculatethat the lower band shown in some samples from whole
liver homogenates or PCs is a product of proteolysis ornonspecific cross-reactivity of the antibody.
Furthermore, Northern blot analysis provided additional evidence of hepatic HSL expression. As shown in
Fig. 3A, the expression of HSL was almost negligible in arefed state, and fasting induced the expression, which indicates that the hepatic expression of HSL is regulated bynutritional status. The expression pattern of HSL might
explain the fact that the hepatic accumulation of CEs in
HSL2/2 mice was more pronounced in the fasted state.
HSL deficiency decreases CE hydrolase activity but not TG
lipase activity in the liver
To further explore the underlying mechanisms, neutral
lipase activities of liver homogenates were measured. In a
dietary cholesterol load experiment (12 h of fasting), CE
hydrolase activity was reduced in HSL2/2 mice (49% reduction in the standard chow group and 53% reductionin the 2% cholesterol chow group; Fig. 4A). In a leptindeficient model, CE hydrolase activity was reduced infasted HSL2/2 mice (31% reduction), and the difference

Fig. 3. Gene expression profile of HSL2/2 mice cross-bred with
Lepob/ob mice. A: Mice were starved for 24 h or refed for 12 h after
a 24 h starvation (female, 16 weeks of age; n 5 4). B: Mice werestarved for 6 h (male, 16 weeks of age; n 5 5). C: HSL2/2 mice
fed a high-cholesterol diet (n 5 5–6; female mice were fed a standard chow diet with or without 2% cholesterol for 7 weeks, andmice were starved for 12 h). Their livers were excised and total
RNA was isolated. Ten micrograms of total RNA from each liver
was pooled and subjected to Northern blotting, followed by hybridization with the indicated cDNA probes. HTGL, hepatic triglyceride lipase; HSL, hormone-sensitive lipase; LDLR, low density
lipoprotein receptor; SRB1, scavenger receptor class B type I.
Loading was normalized by the expression of 36B4 (acidic ribosomal phosphoprotein P0) mRNA, and the fold changes comparedwith the control are displayed above each blot.

between HSL1/1 and HSL2/2 mice was decreased in therefed state, which was in accordance with the expression
pattern. On the other hand, neither leptin nor HSL affected TG lipase activity, and TG lipase activity was increased in the fasted state (Fig. 4C).
Excess cholesterol in the endoplasmic reticulum is esterified by ACAT and stored in cytoplasmic lipid droplets(2). The ACAT activity was not different between HSL1/1
and HSL2/2 mice, and the ACAT activity did not explain
the excess accumulation of CEs in HSL2/2 mice (Fig. 4B).
HSL deficiency alters the expression profiles of genes
controlling liver lipid metabolism
Next, we evaluated the hepatic expression profiles ofgenes regulating lipid homeostasis (Fig. 3). Cholesterol
input through de novo synthesis, represented here by
HMG-CoA synthase, and lipoprotein uptake, represented
by the LDL receptor (1), were decreased in both HSL2/2
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Fig. 2. Immunoblot analysis of HSL protein in the liver. A: Homogenate of liver (50 mg) was subjected to SDS-PAGE in duplicate foreach genotype. In the right lane, homogenate (10 mg) of epididymal
white adipose tissue (WAT) from wild-type male mice was loaded asa control. B: Immunoblot analysis of different types of liver cells
and murine hepatocyte cell lines using polyclonal anti-HSL antibody. Homogenate (20 mg) of each cell population was loaded. In
the right lane, homogenate (4 mg) of epididymal white adipose tissue from wild-type male mice was loaded as a control. PC, parenchymal cell; NPC, nonparenchymal cell; H2.35, H2.35 hepatocyte
cell line; McARH, McARH-7777 hepatoma cell line; MPM, mouse
peritoneal macrophage.

and Lepob/ob/HSL2/2 mice. The reverse cholesterol transport pathway mediated by HDL also transports CE to theliver. The expression levels of two key players in this pathway, hepatic triglyceride lipase and scavenger receptor class
B type I, were not changed.
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DG accumulation was not noticeable in HSL2/2 mice
It has been speculated that HSL is a rate-limiting enzyme in the DG catabolism in adipose tissues, since DG
accumulation and TG lipase activity were observed in adipose tissues of HSL2/2 mice (15, 33). In our study, theacylglycerol content in Lepob/ob/HSL2/2 mice was not significantly different from that in Lepob/ob/HSL1/1 mice.
We performed TLC to examine whether DG was accumulated in the liver of Lepob/ob/HSL2/2 mice. Almost allof the acylglycerol detected in the livers of HSL2/2 mice
consisted of TG, while DG accumulation in adipose tissue was confirmed in both Lep1/1/HSL2/2 and Lepob/ob/
HSL2/2 mice (Fig. 6). It is necessary to further elucidate
whether or not there might be a difference that was undetectable by our methods in the DG content between HSL1/1
and HSL2/2 mice.
CE hydrolysis was impaired in primary cultured hepatocytefrom HSL2/2 mice
HSL is widely expressed in multiple tissues, and HSL
deficiency alters systemic lipid metabolism, including
the impediment of the mobilization of FFA from adipose
tissues and increased remnant lipoprotein clearance via
LPL activity in muscle and white adipose tissue (26). To
eliminate these indirect effects through systemic factors

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Fig. 4. Enzymatic activity of liver homogenates. A, B: HSL2/2 mice
fed a high-cholesterol diet (n 5 5–6; female mice were fed a standard chow diet with or without 2% cholesterol for 7 weeks, andmice were starved for 12 h). C: HSL2/2 mice cross-bred with
Lepob/ob mice. Mice were starved for 24 h or refed for 12 h after
a 24 h starvation (female, 16 weeks of age; n 5 4). A and C
show enzymatic activity of liver homogenates from each genotype. Livers were excised and homogenized in buffer A (250 mM
sucrose, 20 mM Tris-HCl, pH 7.4, and protease inhibitors). S-100
protein was obtained by ultracentrifugation at 100,000 g for
45 min. Buffer exchange to phosphate-buffered saline containing
1 mM EDTA was carried out by dialysis for 4 h. The samples wereincubated at 37°C for 30–60 min in a reaction mixture containingtri[3H]oleoylglycerol or [1-14C]oleate. TGL and CEH denote TG
lipase and CE hydrolase, respectively. For B, microsomal ACAT
activity was measured. Liver microsomal protein was obtained byultracentrifugation at 100,000 g for 45 min. The precipitates were
resuspended and used for assay. ACAT activity in microsomes
was determined by the rate of incorporation of [1-14C]oleoyl-CoA
into the CE fraction. Data are expressed as means 6 SEM. * P ,0.05, ** P , 0.01 as determined by ANOVA followed by the TukeyKramer posthoc test.

HSL deficiency affects the plasma lipoprotein profile
Hepatic lipoprotein secretion is one of the pathways
regulating cholesterol homeostasis in the liver. HSL2/2
mice were reported to have reduced levels of VLDL caused
by decreased FFA mobilization from adipose tissues to theliver (26). The reduction of FFA and VLDL by HSL disruption was also confirmed in the Lepob/ob background(Fig. 5A). Increased plasma HDL-cholesterol in HSL2/2
mice was also reported (26) and confirmed in our study,
but the increase of HDL-cholesterol was blunted in the
Lepob/ob background.
Dietary cholesterol load also altered these lipoprotein
profiles. Plasma total cholesterol was increased by theincreased HDL-cholesterol in HSL2/2 mice fed normal
chow. Measurement of plasma cholesterol levels revealed a significant increase in HSL2/2 mice fed a 2%cholesterol diet (Fig. 5D), and the increase was caused
by increased VLDL/LDL-cholesterol (VLDL-cholesteroland LDL-cholesterol were increased by 92% and 50%,
respectively) (Fig. 5C).
Excessive TG accumulation plays a role in the progression of liver injury. On the other hand, it is not well known
whether CE accumulation leads to liver injury. Therefore,
we examined plasma ALT levels as an indicator of liver
injury. Excessive accumulation of TG induced by leptin
deficiency increased the plasma ALT level, while accumulation of CEs in HSL2/2 mice did not affect plasma ALT(Fig. 5B). In addition, we examined whether histological alteration occurred following the CE accumulation in
Lepob/ob/HSL2/2. Despite the striking increase of CE content in Lepob/ob/HSL2/2 mice, we found no histologicaldifference between Lepob/ob/HSL1/1 and Lepob/ob/HSL2/2
mice (data not shown).

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Fig. 5. Plasma analysis. A, B: HSL2/2 mice cross-bred with Lepob/ob mice (12 weeks of age, male; n 5 10
for each genotype) were starved for 6 h, and blood was collected from the retro-orbital venous plexus. C,
D: HSL2/2 mice fed a high-cholesterol diet [n 5 5–6; female mice (8 weeks) were fed a standard chowdiet with or without 2% cholesterol for 7 weeks, and mice were starved for 12 h]. A and C show analysisof plasma lipid by HPLC. Chol and TG denote cholesterol and triacylglycerol, respectively. For B and D,
plasma alanine aminotransferase (ALT) levels and plasma lipid profiles are shown. TC, total cholesterol.
Data are expressed as means 6 SEM. ** P , 0.01 as determined by ANOVA followed by the Tukey-Kramer
posthoc test.

and reinforce the in vivo results, we also examined cholesterol metabolism in primary cultured hepatocytes. The
CE hydrolase activity in HSL2/2 hepatocytes was reduced
compared with that in HSL1/1 hepatocytes (65% reduction; Fig. 7A). Furthermore, we exposed these cells tohuman LDL (100 mg/ml) and examined [1-14C]oleate incorporation into the CE fraction. Even when the cells

were cultured in medium supplemented with LPDS, theHSL2/2 hepatocytes contained more CE compared with
HSL1/1 hepatocytes (47% increase), although the extent
of CE accumulation was small. In the presence of LDL,
more pronounced CE accumulation was observed (54%increase). These responses were blunted by ACAT inhibitor (10 mM CS-505) (Fig. 7B).
Hormone-sensitive lipase in liver

1835

DISCUSSION
In the present report, we attempted to investigate
whether HSL is involved in the hydrolysis of neutral lipids
in the liver. We show here that HSL was indeed expressedin hepatocytes and that a deficiency of HSL induced hepatic
CE accumulation, especially under two different nutritional
conditions: leptin deficiency and dietary cholesterol load.
To date, several lipases that have CE hydrolase activity
have been identified in hepatocytes, including hepatic
cytosolic CE hydrolase (4), CEL (34, 35), LAL (3), andmicrosomal CE hydrolase (5). In our study, HSL deficiency decreased hepatic CE hydrolase activity and induced hepatic CE accumulation. Among a wide varietyof lipases expressed in hepatocytes, HSL definitely plays
an important role in CE metabolism.
On the other hand, lipases with TG lipase activity
include adipose triglyceride lipase (ATGL) (36), TG hydrolase (37), TG hydrolase-2 (38), hepatic triglyceridelipase (39), LPL, and LAL. Among these lipases, ATGL
1836

Journal of Lipid Research

Volume 49, 2008

would play an important role in hepatic TG metabolism.
ATGL-null mice have been reported to exhibit hepatic
TG accumulation and reduced TG lipase activity (36).
In our study, HSL deficiency affected neither TG lipase
activity nor hepatic TG content. Other TG lipases could
compensate for HSL deficiency, and DG lipase activity
might not be regulated by HSL in hepatocytes, although
it is regulated by HSL in adipocytes.
Liver has two populations of cells that exhibit neutral
CE hydrolase activity: hepatocytes and macrophages. The
expression of HSL in macrophages is widely accepted but
remains controversial to some extent (13, 28–32). The relative contribution of the resident macrophages to the reduced neutral CE hydrolase activity of HSL2/2 mice would
be almost nil because 1) the neutral CE hydrolase activity

Downloaded from www.jlr.org at Univ of Tokyo Medical Library on October 1, 2008

Fig. 6. Lipid separation by TLC. Liver lipid was extracted by thechloroform-methanol method, and aliquots were pooled foreach genotype (n 5 4). Pooled lipids from 0.6 mg of liver tissue
were loaded in each lane. Lipids were separated with hexanediethylether-acetic acid (80:20:1) or chloroform-acetone-acetic
acid (96:4:1) as solvent. Standards are shown at left. As a control,
lipids from epididymal white adipose tissue (WAT) were also separated and visualized. CE, cholesterol ester; DAG, diacylglycerol; FC,free cholesterol; FFA, free fatty acids; MAG, monoacylglycerol; PL,phospholipid; TG, triacylglycerol.

Fig. 7. Primary cultured hepatocytes. Hepatocytes were isolated
from nonfasted HSL2/2 mice and their littermate HSL1/1 miceby the collagenase perfusion method. A: After isolation (male,
10 weeks; n 5 5 for each genotype), the attached cells were maintained in Medium 199 containing Earleʼs salts and 5% fetal calf
serum for 20 h. The medium was replaced with the same medium,and the cells were further incubated for 12 h. The whole cell lysates were assayed for CE hydrolase (CEH) activities. Data are expressed as means 6 SEM. ** P , 0.01 as determined by Studentʼst -test. B: CE formation. Attached cells were incubated in Williamsʼ
E medium containing 5% human LPDS for 48 h and subsequently
incubated in the same medium containing 100 mg/ml LDL and[1-14C]oleate-albumin complex with vehicle or the ACAT inhibitor
CS-505 (10 mM) for 24 h. Intracellular lipids were separated by
TLC, spots corresponding to the CE fraction were scraped, andradioactivities were measured. Data are expressed as means 6
SEM. * P , 0.05, ** P , 0.01 as determined by ANOVA followedby the Tukey-Kramer posthoc test.

might be explained by several factors, including differences in the subcellular localization of lipases and lipid
and the amount of accumulated CEs.
It was reported that hepatic VLDL synthesis is reduced in
HSL2/2 mice as a result of decreased FFA transport fromthe adipose tissue to the liver (26). It was also reported thatthe decreased VLDL-TG in HSL2/2 mice is associated withincreased LPL activity in muscle and white adipose tissue(26). The reduced plasma VLDL in HSL2/2 mice was also
confirmed in our experiments, and these findings were applicable to Lepob/ob/HSL2/2 mice. Despite the reduced VLDL
secretion in HSL2/2 mice, dietary cholesterol load increased
plasma VLDL/LDL-cholesterol. The direct involvementof hepatic HSL in lipoprotein metabolism should be evaluated using primary cultured hepatocytes or liver-specificHSL-deficient mice. As shown in Results, the LDL receptorand HMG-CoA synthase, which are representative genes
regulated by sterol-regulatory element binding protein(SREBP)-2, were downregulated in HSL2/2 mice; therefore,
reduced lipoprotein uptake via the LDL receptor would partially explain these lipoprotein profiles. Upon activation,
SREBP-2 is released from the membrane into the nucleus
as a mature protein by a sequential two-step cleavage process, and the activation is regulated by cellular cholesterol(45). The precise mechanism underlying the transcriptional
regulation in HSL2/2 mice should be further elucidated.
Our experiments revealed that the expression of hepatic
HSL is regulated transcriptionally by nutritional status. It
was reported that the expression of HSL is regulated by
glucose using an adipocyte cell line (46) and that upstream
stimulatory factors 1 and 2 are involved in the transcriptional
regulation of HSL (47). We observed similar transcriptional
regulation in vivo, although the precise mechanisms havenot been elucidated. HSL in adipose tissue is known to playa pivotal role in providing fatty acids to supply energy for
demand in the fasted state. HSL in liver might play a similarrole by hydrolyzing stored CEs and providing fatty acids
for ATP synthesis in the fasted state.
In conclusion, HSL is definitely expressed in hepatocytes, and the deficiency of HSL reduced hepatic CE hydrolase activity but not TG lipase activity. This reductionof CE hydrolase activity induced the accumulation of
CE in liver. This study provides important new insights
into hepatic cholesterol metabolism, especially regarding
hepatic CE hydrolysis.

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