THE JOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 279, No. 15, Issue of April 9, pp. 15084 –15090, 2004
Printed in U.S.A.

Absence of Hormone-sensitive Lipase Inhibits Obesity andAdipogenesis in Lepob/ob Mice*
Received for publication, October 6, 2003, and in revised form, December 28, 2003
Published, JBC Papers in Press, January 28, 2004, DOI 10.1074/jbc.M310985200

Motohiro Sekiya‡, Jun-ichi Osuga‡, Hiroaki Okazaki‡, Naoya Yahagi‡, Kenji Harada§,
Wen-Jun Shen§, Yoshiaki Tamura‡, Sachiko Tomita‡, Yoko Iizuka‡, Ken Ohashi‡,
Mitsuyo Okazaki¶, Masataka Sata储, Ryozo Nagai储, Toshiro Fujita‡, Hitoshi Shimano**,Fredric B. Kraemer§, Nobuhiro Yamada**, and Shun Ishibashi‡ ‡‡§§
From the Departments of ‡Metabolic Diseases and 储Cardiovascular Medicine, Faculty of Medicine, University of Tokyo,
Tokyo 113-8655, Japan, §Veterans Affairs Palo Alto Health Care System and Division of Endocrinology, Stanford
University, Palo Alto, California 94304, the ¶Laboratory of Chemistry, College of Liberal Arts and Sciences, Tokyo
Medical and Dental University, Chiba 272-0827, Japan, **Metabolism, Endocrinology, and Atherosclerosis, Institute ofClinical Medicine, University of Tsukuba, Ibaraki 305-8575, Japan, and the ‡‡Division of Endocrinology and Metabolism,
Department of Medicine, Jichi Medical School, Tochigi 329-0498, Japan

Hormone-sensitive lipase (HSL) plays a crucial role in
the hydrolysis of triacylglycerol and cholesteryl ester invarious tissues including adipose tissues. To explore therole of HSL in the metabolism of fat and carbohydrate,
we have generated mice lacking both leptin and HSL(Lepob/ob/HSLⴚ/ⴚ) by cross-breeding HSLⴚ/ⴚ mice withgenetically obese Lepob/ob mice. Unexpectedly, Lepob/ob/
HSLⴚ/ⴚ mice ate less food, gained less weight, and had
lower adiposity than Lepob/ob/HSLⴙ/ⴙ mice. Lepob/ob/
HSLⴚ/ⴚ mice had massive accumulation of preadipocytes in white adipose tissues with increased expressionof preadipocyte-specific genes (CAAT/enhancer-binding
protein ␤ and adipose differentiation-related protein)and decreased expression of genes characteristic of mature adipocytes (CCAAT/enhancer-binding protein ␣,
peroxisome proliferator activator receptor ␥, and adipocyte determination and differentiation factor 1/sterol
regulatory element-binding protein-1). Consistent withthe reduced food intake, hypothalamic expression ofneuropeptide Y and agouti-related peptide was decreased. Since HSL is expressed in hypothalamus, we
speculate that defective generation of free fatty acids inthe hypothalamus due to the absence of HSL mediates
the altered expression of these orexigenic neuropeptides. Thus, deficiency of both leptin and HSL has unmasked novel roles of HSL in adipogenesis as well as infeeding behavior.

Excessive cellular accumulation of neutral lipids underlies
many diseases such as obesity, type 2 diabetes, and atherosclerosis, all of which are epidemic in industrialized countries.
Therefore, elucidating the metabolic pathways that degrade
excessive neutral lipids is of extreme importance in the preven-* This work was supported in part by grant-in-aid for Scientific Research from the Ministry of Education, Science, and Culture and research grants from Otsuka Pharmaceutical Co., Japan Diabetes Foundation, Takeda Medical Research Foundation, and Asahi LifeFoundation. The costs of publication of this article were defrayed in partby the payment of page charges. This article must therefore be hereby
marked “advertisement” in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
§§ To whom correspondence should be addressed: Division of Endocrinology and Metabolism, Dept. of Medicine, Jichi Medical School,
3311-1 Yakushiji, Minamikawachi-machi, Kawachi-gun, Tochigi 3290498, Japan. Tel.: 81-285-58-7355; Fax: 81-285-40-6035; E-mail:ishibash@jichi.ac.jp.

tion of the diseases caused by lipotoxicity (1).
Hormone-sensitive lipase (HSL)1 is an intracellular neutral
lipase that catalyzes the hydrolysis of cellular triglycerols (TG),diglycerols (DG), monoacylglycerols, and cholesteryl esters aswell as other lipids (2, 3). HSL is expressed in a wide variety oforgans and cells, including adipose tissues, heart, skeletal muscle, adrenal glands, testes, ovaries, and pancreatic ␤-cells, andis under neural and hormonal control.
Several laboratories including ours have generated HSLdeficient (HSL⫺/⫺) mice by targeted gene disruption (4 – 6).
Unexpectedly, these mice showed male sterility due to thefailure of spermatogenesis (4, 7). Although HSL⫺/⫺ mice had adecreased ability to release free fatty acids (FFA) from adipocytes in response to the ␤-adrenergic stimulation both in vivo
and in vitro, they were neither obese nor cold-sensitive (4, 5).
The attenuated development of adipocyte-associated phenotypes may result from the presence of a residual TG lipase
activity in adipocytes, which is induced by both ␤-adrenergic
agonist and tumor necrosis factor-␣ (8). However, it is unknown
how HSL deficiency affects adiposity in the setting of obesity,
which is commonly associated with type 2 diabetes.
Here we show that mice lacking both HSL and leptin(Lepob/ob/HSL⫺/⫺) show impaired adipogenesis and paradoxical resistance to obesity primarily due to the reduced foodintake. These observations in the leptin-deficient state haverevealed new functions of HSL in adipogenesis and feeding
behavior.
EXPERIMENTAL PROCEDURES

Animals—HSL⫺/⫺ mice (4), which were backcrossed five times into
the C57BL/6J background, were intercrossed with mice heterozygous
for leptin deficiency (Lep⫹/ob/C57BL/6J; Jackson Laboratories, Bar
Harbor, ME) to generate double heterozygotes, which were then interbred to produce Lepob/ob/HSL⫺/⫺ mice. Genotyping was performed as
described previously (4, 9). Mice were housed in a temperature-controlled environment with a 12-h light/dark cycle and allowed free accessto water and a standard chow diet (Oriental MF, Oriental Yeast, Tokyo,
Japan). Mice (16 weeks) were sacrificed after a 6-h fast unless otherwise
stated. Mice were sacrificed at the end of the light cycle to isolate
hypothalamic RNA. All experiments were performed in accord withinstitutional guidelines.
Biochemical Analyses—Blood was collected from the retro-orbital
venous plexus after a 6-h fast. Cholesterol (Determiner TC; Kyowa
Medex, Tokyo, Japan), TG and glycerol (TG LH; Wako Chemicals,
1
The abbreviations used are: HSL, hormone-sensitive lipase; TG,
triglycerol(s); DG, diglycerol(s); FFA, free fatty acid(s); WAT, white
adipose tissue; ACTH, adrenocorticotropic hormone.

15084

This paper is available on line at http://www.jbc.org

Hormone-sensitive Lipase Deficiency in Lepob/ob Mice

15085

FIG. 1. HSLⴚ/ⴚ mice are resistant to genetic obesity. a, body weight of each mouse was measured weekly from 4 to 16 weeks (n ⫽ 10). E,
Lep⫹/⫹/HSL⫹/⫹; 䡺, Lep⫹/⫹/HSL⫹/⫺; 䡺, Lep⫹/⫹/HSL⫺/⫺; ●, Lepob/ob/HSL⫹/⫹; Œ, Lepob/ob/HSL⫹/⫺; f, Lepob/ob/HSL⫺/⫺. b, tissue weights of eachmouse were measured at 16 weeks of age after a 6-h fast (n ⫽ 10). L, liver; B, brown adipose tissue; H, heart; P, pancreas; Pg, paragonadal WAT;
Pr, perirenal WAT; Sc, subcutaneous WAT; Q, quadriceps; EDL, extensor digitorum longus. Each value represents the mean ⫾ S.E. * and **,significance at p ⬍ 0.05 and p ⬍ 0.01, respectively, versus HSL⫹/⫹ mice in the same Lep background.
Tokyo, Japan), and FFA (NEFA C; Wako Chemicals) were measured
enzymatically. Plasma glucose was measured by ANTSENSE II (Bayer
Medical, Tokyo, Japan); plasma insulin was measured by the mouseinsulin ELISA kit (Morinaga, Tokyo, Japan); and plasma corticosterone
was measured by the EIA kit (Diagnostic Systems Laboratories, Inc.,
Webster, TX).
Immunoblot Analysis—Hypothalamic regions were excised from
8-month-old male mice, homogenized in 0.25 M sucrose buffer (20 mM
Tris-HCl, pH 7.4, 0.25 M sucrose, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 5 ␮g/ml leupeptin, 2.8 ␮g/ml aprotinin), and centrifuged at 15,000 rpm for 15 min. Fifty ␮g of the supernatant fraction was
subjected to SDS/PAGE analysis. S100 protein (10 ␮g) from BAT of wild
type mouse was used as a positive control. Immunoblot analysis was
performed using the ECL kit (Amersham Biosciences) as described
previously (4).
Northern Blot Analysis—Total RNA was isolated by Trizol reagent(Invitrogen). Hypothalamic RNA was isolated as described previously(10). Five ␮g of hypothalamic RNA from each mouse or 7.5–10 ␮g ofwhite adipose tissue (WAT) RNA equally pooled from 3–5 mice of eachgenotype were electrophoresed in a 1% agarose gel containing formaldehyde and transferred to a nylon membrane. The membranes were
hybridized with probes, which were labeled with [␣-32P]dCTP using the
Megaprime DNA labeling system kit (Amersham Biosciences) in Rapidhyb Buffer (Amersham Biosciences) or ULTRAhyb hybridization buffer(Ambion, Austin, TX), and analyzed by a BAS2000 bioimaging analyzer(Fuji Photo Film, Tokyo, Japan). Loading was normalized by the expression of 36B4.
Histology—Sixteen-week-old mice were sacrificed by decapitation.
Tissues were excised, fixed in 10% neutral buffered formalin, embedded
in paraffin, and stained with hematoxylin-eosin. Cell size was estimated by NIH Image. After blocking with 0.5% goat serum, sections of
epididymal fat pads were incubated with anti-mouse S-100 antibody(Sigma) overnight and were incubated with an anti-rabbit secondary

antibody followed by staining with the avidin-biotin complex-alkaline
phosphatase method and counterstaining with Harris’s hematoxylin.
Apoptotic cells were detected by terminal deoxynucleotidyltransferasemediated dUTP nick end-labeling using an in situ apoptosis detection
kit (Takara Biomedicals Otsu, Japan) according to the manufacturer’s
instructions, with counterstaining with methyl green (Wako Chemicals). Proliferating cells were detected by bromodeoxyuridine immunostaining. Two h before sacrifice, bromodeoxyuridine (Sigma) was administered intraperitoneally at 50 mg/kg body weight in phosphatebuffered saline. After fixation with 10% neutral buffered formalin,mouse epididymal fat was embedded in paraffin. Immunostaining was
performed by a bromodeoxyuridine In-Situ Detection Kit (PharMingen,
San Diego, CA) and counterstained with Mayer’s hematoxylin (Wako
Chemicals).
Detection of DNA Ladder—Adipose tissue DNA was extracted as
described previously (4). Five ␮g of DNA end-labeled with [␣-32P]dCTP
by Klenow was subjected to electrophoresis in a 1.5% agarose gel andthen transferred to nylon membranes, as described previously (11). As
a control, mouse thymocyte apoptotic DNA was loaded. In brief, mouse
thymus was ground over a mesh, and the isolated thymocytes were
induced to undergo apoptosis by culturing in RPMI containing 10% calf
serum and 1 ␮M dexamethasone at 37 °C for 6 h.
Food Intake and Body Temperature—Food intake and body weight
were measured for 3 days after the mice were individually housed andadapted for 1 week. Feeding efficiency was defined as an increase inbody weight divided by food intake during 3 days (n ⫽ 8 –10). Body
temperature was measured by an NK-YSI precision N550 thermometerand its probe (Nikkiso-YSI, Japan). These experiments were performedat the end of light cycle at the age of 14 weeks.
Oxygen Consumption—After a 1-day acclimation period, oxygen andbicarbonate expired by mice (14 weeks old) were measured every 7 minfor 24 h by a calorimetric system (Alco System model, Chiba Japan).
Oxygen consumption was normalized by body weight raised to the 0.7

15086

Hormone-sensitive Lipase Deficiency in Lepob/ob Mice

FIG. 2. Morphological analysis of epididymal WAT. Mice were sacrificed after a 6-h fast at the age of 16 weeks. a, sections of epididymal
WAT were stained with hematoxylin-eosin. b, cell number is plotted against log of cytoplasmic area. c, immunohistochemistry of S100 protein.
power, which is proportional to body surface area.
Statistics—Statistical differences between groups were analyzed by
one-way analysis of variance and a post hoc Tukey-Kramer test, unlessotherwise stated.
RESULTSHSL Deficiency Ameliorates Obesity in Lepob/ob Background—In the Lep⫹/⫹ background, there was no difference inbody weight among HSL⫹/⫹, HSL⫹/⫺, and HSL⫺/⫺ mice on anormal chow diet (Fig. 1a). In the Lepob/ob background, however, body weight of doubly homozygous mice was reduced by
26% compared with the Lepob/ob/HSL⫺/⫺ mice at the age of 16
weeks (Fig. 1a). The difference was discernible at the age of 11
weeks in males and 9 weeks in females. Furthermore, theage-dependent weight gain was negligible in Lepob/ob/HSL⫺/⫺mice after 10 weeks of age.
At the age of 16 weeks, the weight of WAT in three different
regions was selectively reduced in Lepob/ob/HSL⫺/⫺ mice compared with Lepob/ob/HSL⫹/⫹ mice; paragonadal, perirenal,and subcutaneous WAT were reduced by 58, 51, and 44%,
respectively (Fig. 1b).
HSL⫺/⫺ Mice Have an Increased Number of Preadipocytelike Cells in WAT—In the Lep⫹/⫹ background, WAT from
HSL⫺/⫺ mice contained clusters of small cells devoid of lipids inaddition to lipid-filled mature adipocytes (Fig. 2a), as we reported previously (4). In the Lepob/ob background, the small
cells, which appeared slightly increased in Lepob/ob/HSL⫹/⫹
mice, were robustly increased in Lepob/ob/HSL⫺/⫺ mice (Fig. 2,
a and b).
To determine the identity of these small cells, we performed
immunohistochemistry for S-100 protein, which is used as amarker of adipocytes (12), particularly of preadipocytes, andneural cells (Fig. 2c). The small cells were strongly positive for
S-100, indicating that they were preadipocytes. Upon Northernblot analyses of WAT (Fig. 3a), the expression of adipose differentiation-related protein (13) and CAAT/enhancer-binding
protein ␤, which is dominant during the early stage of differentiation of adipocytes (14), was increased in HSL⫺/⫺ WAT
compared with HSL⫹/⫹ WAT in both Lep⫹/⫹ and Lepob/ob backgrounds. On the other hand, the expression of CAAT/enhancerbinding protein ␣, which is dominant during the late stage ofdifferentiation (14), adipocyte determination and differentiation factor 1/sterol regulatory element-binding protein-1 (15),fatty acid synthase (16), and peroxisome proliferator activator
receptor ␥ (17) was decreased in HSL⫺/⫺ WAT compared with
HSL⫹/⫹ WAT in both Lep⫹/⫹ and Lepob/ob backgrounds. These
results strongly support the results of immunohistochemistry
and the conclusion that preadipocytes were increased in
HSL⫺/⫺ WAT. No significant differences were observed in theexpression of lipoprotein lipase and uncoupling protein-2 between Lepob/ob/HSL⫹/⫹ and Lepob/ob/HSL⫺/⫺ mice.
To rule out the possibility that the small cells are apoptotic
due to overaccumulation of intracellular TG, we performed
terminal deoxynucleotidyltransferase-mediated dUTP nickend-labeling staining and DNA ladder detection. Although
Lepob/ob WAT contained an increased number of terminal deoxynucleotidyltransferase-mediated dUTP nick end labelingpositive apoptotic cells and increased DNA ladder formationcompared with Lep⫹/⫹ WAT, there was no difference between
HSL⫹/⫹ and HSL⫺/⫺ WAT (Fig. 3b). Furthermore, we performed in vivo bromodeoxyuridine labeling to determineHormone-sensitive Lipase Deficiency in Lepob/ob Mice

FIG. 3. Northern blot analysis and DNA ladder of epididymal
WAT. a, Northern blot analysis. Total RNA was isolated and pooled
equally from three mice. A cDNA probe for 36B4 (acidic ribosomal
phosphoprotein P0 (ADRP)) was used as a loading control. The numbersin the figure represent relative intensities of the bands compared withthose of Lep⫹/⫹/HSL⫹/⫹. b, DNA ladder. Left lane, DNA from mousethymocyte that had been treated with dexamethasone. Labeled DNA (5␮g) was loaded in each lane. TNF␣, tumor necrosis factor ␣. C/EBP,
CAAT/enhancer-binding protein; PPAR, peroxisome proliferator activator receptor.

whether the small cells have increased proliferative activity.
No increase in bromodeoxyuridine-positive cells was observedin HSL⫺/⫺ WAT (data not shown).
Food Intake Is Reduced in Obese HSL⫺/⫺ Mice—To determine the causes of the lower adiposity in Lepob/ob/HSL⫺/⫺
mice, we examined food intake. In the Lep⫹/⫹ background,
there was no difference in food intake between HSL⫹/⫹ andHSL⫺/⫺ mice. In the Lepob/ob background, food intake, which

15087

FIG. 4. Food intake and plasma corticosterone levels. a, foodintake was measured at the age of 14 weeks (n ⫽ 10). b, feeding
efficiency was determined at the age of 14 weeks (n ⫽ 8 –10). c, basal
plasma corticosterone levels at the age of 9 weeks (n ⫽ 10).

was increased compared with the Lep⫹/⫹ background, was reduced by 19% in HSL⫺/⫺ mice compared with HSL⫹/⫹ mice at
the age of 14 weeks (Fig. 4a). The reduced food intake wasapparent at least after 8 weeks of age (data not shown).
Lepob/ob/HSL⫺/⫺ mice had significantly lower feeding efficiency
than Lepob/ob/HSL⫹/⫹ mice (Fig. 4b), indicating that the reduced food intake was not the only mechanism contributing to
the leanness of Lepob/ob/HSL⫺/⫺ mice.
Adrenalectomy reduces food intake (18, 19) in Lepob/ob mice,
which are in hyper-ACTH status, and HSL deficiency reduces
the corticosterone response to ACTH (20, 21). Therefore, wemeasured basal plasma corticosterone levels to examine
whether adrenal insufficiency underlies the reduced food intake. There was no significant decrease in the plasma corticos-

15088

Hormone-sensitive Lipase Deficiency in Lepob/ob Mice

FIG. 6. Body temperature and oxygen consumption. a, core bodytemperature was measured at the age of 14 weeks (n ⫽ 10). b, oxygen
consumption was measured under a 12-h light/dark cycle and freeaccess to food and water at the age of 12 weeks (n ⫽ 8 –9). Oxygen
consumption was normalized by body surface area. E, Lep⫹/⫹/HSL⫹/⫹;䡺, Lep⫹/⫹/HSL⫺/⫺; ●, Lepob/ob/HSL⫹/⫹; f, Lepob/ob/HSL⫺/⫺. Each value
represents the mean ⫾ S.E.

FIG. 5. Hypothalamic expression of HSL and genes encoding
orexigenic and anorexigenic neuropeptides and fatty acid synthase (FAS). a, Western blot analysis of HSL protein in the hypothalamic regions of either Lep⫹/⫹/HSL⫹/⫹ or Lep⫹/⫹/HSL⫺/⫺ mice. Wildtype brown adipose tissue (BAT) was used as a positive control. b,
Northern blot analyses of the hypothalamic genes. Mice aged 16 weeks
were sacrificed at the end of light cycle. RNA from three mice was
loaded in each lane. 36B4 (acidic ribosomal phosphoprotein P0) wasused as a loading control. c, the radioactivity was measured by the
PhosphorImager and statistically analyzed. Each value represents themean ⫾ S.E. POMC, pro-opiomelanocortin.

terone levels in Lepob/ob/HSL⫺/⫺ mice compared with Lepob/ob/
HSL⫹/⫹ mice (Fig. 4c).
Hypothalamus expressed HSL protein in wild-type mice but
not in HSL⫺/⫺ mice (Fig. 5a). The suppressed food intake in
Lepob/ob mice led us to examine the expression of various neuropeptides that govern appetite and satiety in the hypothalamus (Fig. 5, b and c). The expression levels of NPY and AgRP,
both of which were induced in Lepob/ob background, were de-creased nearly to the level of lean Lep⫹/⫹ mice (Fig. 5, b and c).
The expression levels of pro-opiomelanocortin, which were suppressed in the Lepob/ob background, were not different between
HSL⫹/⫹ and HSL⫺/⫺ mice (Fig. 5, b and c).
In addition to food intake, energy expenditure determines
adiposity. Body temperature, which was reduced in the
Lepob/ob background, was not significantly different between
HSL⫹/⫹ and HSL⫺/⫺ mice (Fig. 6a). Consistently, the mean
oxygen consumption during 24 h was not significantly different
between HSL⫹/⫹ and HSL⫺/⫺ mice in the Lepob/ob backgroundwhen normalized by body surface area (males, 0.120 ⫾ 0.024
versus 0.136 ⫾ 0.019 ml/min/g0.7, p ⫽ 0.18; females, 0.119 ⫾0.021 versus 0.135 ⫾ 0.025 ml/min/g0.7, p ⫽ 0.18), although
Lepob/ob/HSL⫺/⫺ mice appeared to have higher oxygen consumption than Lepob/ob/HSL⫹/⫹ mice (Fig. 6b).
HSL Deficiency Decreases Plasma Levels of FFA and
Glycerol—Lepob/ob/HSL⫺/⫺ mice showed significant reductionin plasma levels of FFA and glycerol compared with Lepob/ob/
HSL⫹/⫹ mice (FFA, 663 ⫾ 67 versus 1,174 ⫾ 17 ␮M, p ⬍ 0.01;
glycerol, 10.0 ⫾ 2.8 versus 43.4 ⫾ 6.0 mg/dl, p ⬍ 0.01). In thelean Lep⫹/⫹/HSL⫺/⫺ mice, plasma cholesterol levels were elevated, whereas plasma TG levels were decreased comparedwith those in Lep⫹/⫹/HSL⫹/⫹ mice, which was due to an increase in high density lipoproteins and a decrease in very low
density lipoproteins, respectively (data not shown), as reported
previously (22). In the Lepob/ob background, no significant difference was observed between HSL⫹/⫹ and HSL⫺/⫺ mice.
Leanness and low plasma FFA levels are commonly associ-Hormone-sensitive Lipase Deficiency in Lepob/ob Mice
ated with increased insulin sensitivity, whereas impaired adipogenesis is associated with insulin resistance (23). To estimate insulin sensitivity, we measured blood levels of glucose
and insulin. There were no differences in the blood glucose andplasma insulin levels between Lepob/ob/HSL⫹/⫹ and Lepob/ob/
HSL⫺/⫺ mice when fasted (data not shown), indicating that
insulin sensitivity was not significantly different between
them.
DISCUSSION

In the present studies, we use Lepob/ob/HSL⫺/⫺ mice to showthat the absence of HSL inhibits adipogenesis and feeding
behavior, thereby decreasing adiposity in leptin-deficient obesity. Resistance to obesity was also observed in a high fat
diet-induced obesity model (24).
Adipocytes in WAT of Lepob/ob/HSL⫺/⫺ mice displayed exaggerated size heterogeneity (Fig. 2, a and b). In particular, small
cells were remarkably increased. Although adipocytes from
Lepob/ob WAT are prone to apoptosis upon insulin depletion(25) and indeed Lepob/ob WAT contained a substantial number
of apoptotic cells, HSL deficiency did not further increase thenumber of apoptotic cells in either the Lep⫹/⫹ or Lepob/ob settings (Fig. 3c). Since the majority of the small cells were positive for S-100 protein (Fig. 2c), we consider the small cells to bepreadipocytes. This view is consistent with the gene expression
profiles of WAT; adipose differentiation-related protein and
CAAT/enhancer-binding protein ␤, which characterize theearly phase of adipocyte differentiation, were up-regulated,
and CAAT/enhancer-binding protein ␣, peroxisome proliferator
activator receptor ␥, adipocyte determination and differentiation factor 1/sterol regulatory element-binding protein-1, andfatty acid synthase, which characterize the mature stage ofadipocyte differentiation, were down-regulated (Fig. 3a). Basedon these results, we speculate that HSL deficiency inhibits
adipogenesis in a certain population of preadipocytes, thereby
stimulating their accumulation. Substances released from adipocytes of Lepob/ob/HSL⫺/⫺ mice may mediate the inhibition of
adipocyte differentiation. In this context, it is of note that FFA,
whose production was reduced in HSL⫺/⫺ mice, induces differentiation of preadipocytes (26 –29). Moreover, Janke et al. (30)have recently reported that angiotensin II secreted by matureadipocytes inhibits differentiation of preadipocytes in culture.
It is also possible that intracellular lipids such as DG, which
are accumulated in the absence of HSL (6), directly inhibit
adipogenesis. It is tempting to speculate that protein kinase C
activation by DG inhibits glycogen synthase kinase-3 (31),thereby enhancing signaling of Wnt, a negative regulator of
adipogenesis (32). The impaired adipogenesis observed in
Lepob/ob/HSL⫺/⫺ mice appears to be inconsistent with our previous findings that HSL⫺/⫺ embryonic fibroblasts are indistinguishable from wild-type embryonic fibroblasts in adipogenic
ability (8). In the experiments in vitro, a pharmacological doseof insulin, methyl-isobutylxanthine, or dexamethasone in the
differentiation induction medium may have overcome the reduced adipogenic potential of HSL⫺/⫺ embryonic fibroblasts.
Indeed, it was reported that exposure to methyl-isobutylxanthine or cAMP is sufficient to suppress expression of Wnt10b in
3T3-L1 cells (33).
Hyperphagia of leptin-deficient Lepob/ob is mediated by the
up-regulation of the orexigenic neuropeptides, NPY (34) and
AgRP (35), with concomitant suppression of the pro-opiomelanocortin gene, which encodes the anorexigenic ␣-melanocytestimulating hormone (36). Since disruption of NPY (37) or its
cognate receptor (Y2 receptor) (38) attenuates the obesity of
Lepob/ob mice, the reduced expression of NPY and possibly
AgRP may account for the decreased food intake in Lepob/ob/
HSL⫺/⫺ mice (Fig. 5, b and c). Together with the hypothalamic

15089

expression of HSL (Fig. 5a), these results strongly indicate thecrucial role of hypothalamic metabolism of fatty acids in thecontrol of feeding behavior. In this context, it is noteworthy
that inhibition of FFA synthesis by fatty acid synthase inhibitors shows similar suppressing effects on food intake (39) as
well as on fasting-induced increase in the hypothalamic expression of NPY and AgRP (40). Likewise, inhibition of hypothalamic carnitine palmitoyltransferase-1 decreases food intake(41). Neither body temperature nor oxygen consumption was
different between Lepob/ob/HSL⫺/⫺ and Lepob/ob/HSL⫹/⫹ mice
(Fig. 6). Thus, the reduced food intake as well as the defective
differentiation of preadipocytes may account for the lower adipocity in Lepob/ob/HSL⫺/⫺ mice at least in mice aged older than
8 weeks (Fig. 1). It is interesting to note that the lower adiposity was not observed in mice younger than 8 weeks. We speculate that non-HSL TG lipase compensates for the deficiency of
HSL until about 8 weeks of age in terms of growth andadiposity.
Leanness or resistance to obesity has been reported in othermurine models with mutated genes that are instrumental in
fatty acid metabolism. For example, mice lacking the RII␤subunit of protein kinase A (42) or perilipin (43) have increased
lipolysis; mice lacking acyl-CoA:diacylglycerol transferase (44)or mitochondrial glycerol-3-phosphate acyltransferase (45)have reduced synthesis of TG. In particular, mice with amutation in the peroxisome proliferator activator receptor
␥-binding site in the gene for the cytosolic form of phosphoenolpyruvate carboxykinase, which lack expression of phosphoenolpyruvate carboxykinase activity in WAT, are similar to
Lepob/ob/HSL⫺/⫺ mice in that their WAT also has accumulationof lipodystrophy-like small cells dispersed among normal large
fat cells (46). The significance of this similarity is currently
unknown.
In conclusion, Lepob/ob/HSL⫺/⫺ mice have impaired adipogenesis and resistance to obesity at least partially due to the
reduced food intake. These findings should provide the basis forunderstanding the pathophysiology of obesity and can be exploited to develop novel therapy for the endemic disease.
REFERENCES
1. Unger, R. H. (2002) Annu. Rev. Med. 53, 319 –336
2. Holm, C., Osterlund, T., Laurell, H., and Contreras, J. A. (2000) Annu. Rev.
Nutr. 20, 365–393
3. Kraemer, F. B., and Shen, W. J. (2002) J. Lipid Res. 43, 1585–1594
4. Osuga, J., Ishibashi, S., Oka, T., Yagyu, H., Tozawa, R., Fujimoto, A., Shionoiri, F., Yahagi, N., Kraemer, F. B., Tsutsumi, O., and Yamada, N. (2000)
Proc. Natl. Acad. Sci. U. S. A. 97, 787–792
5. Wang, S. P., Laurin, N., Himms-Hagen, J., Rudnicki, M. A., Levy, E., Robert,
M. F., Pan, L., Oligny, L., and Mitchell, G. A. (2001) Obes. Res. 9, 119 –128
6. Haemmerle, G., Zimmermann, R., Hayn, M., Theussl, C., Waeg, G., Wagner,E., Sattler, W., Magin, T. M., Wagner, E. F., and Zechner, R. (2002) J. Biol.
Chem. 277, 4806 – 4815
7. Chung, S., Wang, S. P., Pan, L., Mitchell, G., Trasler, J., and Hermo, L. (2001)
Endocrinology 142, 4272– 4281
8. Okazaki, H., Osuga, J., Tamura, Y., Yahagi, N., Tomita, S., Shionoiri, F.,
Iizuka, Y., Ohashi, K., Harada, K., Kimura, S., Gotoda, T., Shimano, H.,
Yamada, N., and Ishibashi, S. (2002) Diabetes 51, 3368 –3375
9. Yahagi, N., Shimano, H., Hasty, A. H., Matsuzaka, T., Ide, T., Yoshikawa, T.,
Amemiya-Kudo, M., Tomita, S., Okazaki, H., Tamura, Y., Iizuka, Y.,
Ohashi, K., Osuga, J., Harada, K., Gotoda, T., Nagai, R., Ishibashi, S., andYamada, N. (2002) J. Biol. Chem. 277, 19353–19357
10. Kumar, M. V., Shimokawa, T., Nagy, T. R., and Lane, M. D. (2002) Proc. Natl.
Acad. Sci. U. S. A. 99, 1921–1925
11. Dix, D. J., Allen, J. W., Collins, B. W., Mori, C., Nakamura, N., Poorman-Allen,P., Goulding, E. H., and Eddy, E. M. (1996) Proc. Natl. Acad. Sci. U. S. A.
93, 3264 –3268
12. Cinti, S., Cigolini, M., Morroni, M., and Zingaretti, M. C. (1989) Anat. Rec. 224,
466 – 472
13. Jiang, H. P., and Serrero, G. (1992) Proc. Natl. Acad. Sci. U. S. A. 89,7856 –7860
14. Darlington, G. J., Ross, S. E., and MacDougald, O. A. (1998) J. Biol. Chem. 273,
30057–30060
15. Tontonoz, P., Kim, J. B., Graves, R. A., and Spiegelman, B. M. (1993) Mol. Cell.
Biol. 13, 4753– 4759
16. Mackall, J. C., Student, A. K., Polakis, S. E., and Lane, M. D. (1976) J. Biol.
Chem. 251, 6462– 6464
17. Chawla, A., Schwarz, E. J., Dimaculangan, D. D., and Lazar, M. A. (1994)
Endocrinology 135, 798 – 800

15090Hormone-sensitive Lipase Deficiency in Lepob/ob Mice

18. Saito, M., and Bray, G. A. (1984) Am. J. Physiol. 246(1 Pt 2), R20 –5
19. Makimura, H., Mizuno, T. M., Roberts, J., Silverstein, J., Beasley, J., andMobbs, C. V. (2000) Diabetes 49, 1917–1923
20. Kraemer, F. B., Shen, W. J., Natu, V., Patel, S., Osuga, J., Ishibashi, S., andAzhar, S. (2002) Endocrinology 143, 801– 806
21. Li, H., Brochu, M., Wang, S. P., Rochdi, L., Cote, M., Mitchell, G., and GalloPayet, N. (2002) Endocrinology 143, 3333–3340
22. Haemmerle, G., Zimmermann, R., Strauss, J. G., Kratky, D., Riederer, M.,
Knipping, G., and Zechner, R. (2002) J. Biol. Chem. 277, 12946 –12952
23. Shimomura, I., Hammer, R. E., Richardson, J. A., Ikemoto, S., Bashmakov, Y.,
Goldstein, J. L., and Brown, M. S. (1998) Genes Dev. 12, 3182–3194
24. Harada, K., Shen, W. J., Patel, S., Natu, V., Wang, J., Osuga, J. I., Ishibashi,S., and Kraemer, F. B. (2003) Am. J. Physiol. Endocrinol. Metab. 285,
E1182–E1195
25. Loftus, T. M., Kuhajda, F. P., and Lane, M. D. (1998) Proc. Natl. Acad. Sci.
U. S. A. 95, 14168 –14172
26. Amri, E. Z., Bertrand, B., Ailhaud, G., and Grimaldi, P. (1991) J. Lipid Res. 32,
1449 –1456
27. Amri, E. Z., Ailhaud, G., and Grimaldi, P. (1991) J. Lipid Res. 32, 1457–1463
28. Grimaldi, P. A., Knobel, S. M., Whitesell, R. R., and Abumrad, N. A. (1992)
Proc. Natl. Acad. Sci. U. S. A. 89, 10930 –10934
29. Amri, E. Z., Ailhaud, G., and Grimaldi, P. A. (1994) J. Lipid Res. 35, 930 –937
30. Janke, J., Engeli, S., Gorzelniak, K., Luft, F. C., and Sharma, A. M. (2002)
Diabetes 51, 1699 –1707
31. Goode, N., Hughes, K., Woodgett, J. R., and Parker, P. J. (1992) J. Biol. Chem.
267, 16878 –16882
32. Ross, S. E., Hemati, N., Longo, K. A., Bennett, C. N., Lucas, P. C., Erickson,
R. L., and MacDougald, O. A. (2000) Science 289, 950 –953
33. Bennett, C. N., Ross, S. E., Longo, K. A., Bajnok, L., Hemati, N., Johnson,
K. W., Harrison, S. D., and MacDougald, O. A. (2002) J. Biol. Chem. 277,
30998 –31004

34. Wilding, J. P., Gilbey, S. G., Bailey, C. J., Batt, R. A., Williams, G., Ghatei,
M. A., and Bloom, S. R. (1993) Endocrinology 132, 1939 –1944
35. Shutter, J. R., Graham, M., Kinsey, A. C., Scully, S., Luthy, R., and Stark, K. L.
(1997) Genes Dev. 11, 593– 602
36. Thornton, J. E., Cheung, C. C., Clifton, D. K., and Steiner, R. A. (1997)
Endocrinology 138, 5063–5066
37. Erickson, J. C., Hollopeter, G., and Palmiter, R. D. (1996) Science 274,
1704 –1707
38. Sainsbury, A., Schwarzer, C., Couzens, M., and Herzog, H. (2002) Diabetes 51,
3420 –3427
39. Loftus, T. M., Jaworsky, D. E., Frehywot, G. L., Townsend, C. A., Ronnett,
G. V., Lane, M. D., and Kuhajda, F. P. (2000) Science 288, 2379 –2381
40. Shimokawa, T., Kumar, M. V., and Lane, M. D. (2002) Proc. Natl. Acad. Sci.
U. S. A. 99, 66 –71
41. Obici, S., Feng, Z., Arduini, A., Conti, R., and Rossetti, L. (2003) Nat. Med. 9,756 –761
42. Cummings, D. E., Brandon, E. P., Planas, J. V., Motamed, K., Idzerda, R. L.,
and McKnight, G. S. (1996) Nature 382, 622– 626
43. Martinez-Botas, J., Anderson, J. B., Tessier, D., Lapillonne, A., Chang, B. H.,
Quast, M. J., Gorenstein, D., Chen, K. H., and Chan, L. (2000) Nat. Genet.
26, 474 – 479
44. Smith, S. J., Cases, S., Jensen, D. R., Chen, H. C., Sande, E., Tow, B., Sanan,
D. A., Raber, J., Eckel, R. H., and Farese, R. V., Jr. (2000) Nat. Genet. 25,
87–90
45. Hammond, L. E., Gallagher, P. A., Wang, S., Hiller, S., Kluckman, K. D.,
Posey-Marcos, E. L., Maeda, N., and Coleman, R. A. (2002) Mol. Cell. Biol.
22, 8204 – 8214
46. Olswang, Y., Cohen, H., Papo, O., Cassuto, H., Croniger, C. M., Hakimi, P.,
Tilghman, S. M., Hanson, R. W., and Reshef, L. (2002) Proc. Natl. Acad. Sci.
U. S. A. 99, 625– 630