Am J Physiol Endocrinol Metab 285: E1182–E1195, 2003.
First published September 3, 2003; 10.1152/ajpendo.00259.2003.

Resistance to high-fat diet-induced obesity and alteredexpression of adipose-specific genes in HSL-deficient mice
Kenji Harada,1,2 Wen-Jun Shen,1,2 Shailja Patel,1 Vanita Natu,1 Jining Wang,1,2
Jun-ichi Osuga,3 Shun Ishibashi,4 and Fredric B. Kraemer1,21Veterans Affairs Palo Alto Health Care System, Palo Alto 94304; 2Department of Medicine, Stanford
University, Stanford, California 94305; 3Department of Metabolic Diseases, University of Tokyo,
Tokyo 113-8655; and 4Department of Internal Medicine, Jichi Medical School, Tochigi 329-0498, Japan
Submitted 11 June 2003; accepted in final form 21 August 2003

Harada, Kenji, Wen-Jun Shen, Shailja Patel, VanitaNatu, Jining Wang, Jun-ichi Osuga, Shun Ishibashi,
and Fredric B. Kraemer. Resistance to high-fat diet-induced obesity and altered expression of adipose-specificgenes in HSL-deficient mice. Am J Physiol Endocrinol Metab
285: E1182–E1195, 2003. First published September 3, 2003;
10.1152/ajpendo.00259.2003.—To elucidate the role of hormone-sensitive lipase (HSL) in diet-induced obesity, HSLdeficient (HSL⫺/⫺) and wild-type mice were fed normal chowor high-fat diets. HSL⫺/⫺ mice were resistant to diet-induced
obesity showing higher core body temperatures. Weight andtriacylglycerol contents were decreased in white adipose tissue (WAT) but increased in both brown adipose tissue (BAT)
and liver of HSL⫺/⫺ mice. Serum insulin levels in the fed
state and tumor necrosis factor-␣ mRNA levels in adipose
tissues were higher, whereas serum levels of adipocyte complement-related protein of 30 kDa (ACRP30)/adiponectin andleptin, as well as mRNA levels of ACRP30/adiponectin, leptin, resistin, and adipsin in WAT, were lower in HSL⫺/⫺mice than in controls. Expression of transcription factors
associated with adipogenesis (peroxisome proliferator-activated receptor-␥, CAAT/enhancer-binding protein-␣) and lipogenesis (carbohydrate response element-binding protein,adipocyte determination- and differentiation-dependent factor-1/sterol regulatory element-binding protein-1c), as well
as of adipose differentiation markers (adipocyte lipid-binding
protein, perilipin, lipoprotein lipase), lipogenic enzymes(glycerol-3-phosphate acyltransferase, acyl-CoA:diacylglycerol acyltransferase-1 and -2, fatty acid synthase, ATP citrate lyase) and insulin signaling proteins (insulin receptor,
insulin receptor substrate-1, GLUT4), was suppressed in
WAT but not in BAT of HSL⫺/⫺ mice. In contrast, expressionof genes associated with cholesterol metabolism (sterol-regulatory element-binding protein-2, 3-hydroxy-3-methylglutaryl-CoA reductase, acyl-CoA:cholesterol acyltransferase-1)
and thermogenesis (uncoupling protein-2) was upregulated
in both WAT and BAT of HSL⫺/⫺ mice. Our results suggestthat impaired lipolysis in HSL deficiency affects lipid metabolism through alterations of adipose differentiation and adipose-derived hormone levels.
adipocyte; differentiation; insulin; leptin; fatty liver

HORMONE-SENSITIVE LIPASE (HSL) mediates the cytosolichydrolysis of triacylglycerols (lipolysis) and cholesteryl

Address for reprint requests and other correspondence: F. B.
Kraemer, Div. of Endocrinology, S-025, Stanford University, Stanford, CA 94305–5103 (E-mail: fbk@stanford.edu).
E1182

esters (12). HSL is expressed in various tissues, including white (WAT) and brown adipose tissues (BAT),
cardiomyocytes, adrenocortical cells, and gonads (11).
Because HSL is responsible for the release of free fatty
acids (FFA) from stored triacylglycerols in adipose tissues, the enzyme has been proposed to play an essential role in the regulation of body weight and fat mass.
We previously reported, however, that the body weightof HSL-deficient (HSL⫺/⫺) mice generated by homologous recombination fed a normal chow diet did not
differ from that of wild-type (HSL⫹/⫹) mice despite thepresence of a markedly suppressed hydrolysis of triacylglycerols and cholesteryl esters in adipocytes. Moreover, the weights of white adipose tissues showed atendency to be lower in HSL⫺/⫺ mice than in HSL⫹/⫹mice (27).
A combination of genetic and environmental factors
causes obesity, which is associated with disorders suchas glucose intolerance, diabetes, hyperlipidemia, hypertension, and atherosclerosis. Among the environmental factors, intake of high-fat food is well known tocontribute to the development of obesity.
Adipose tissues are not static sites for lipid storage
but play active roles in the regulation of lipid andcarbohydrate metabolism. Adipocytes are the source ofseveral hormonal factors (18, 20, 21, 38, 39). Adipose
differentiation (adipogenesis) is regulated by a series ofinteractions between specific transcription factors,
which result in the expression of adipose-specific proteins (adipose markers) and enzymes for triglyceride
synthesis (lipogenesis) as well as insulin-signaling
pathways (22, 30, 36). Furthermore, BAT of rodents
constitutes the major organ of thermogenesis, which is
responsible for energy expenditure (14).
In the present study, we have sought to elucidate therole of HSL in diet-induced obesity by comparing the
responses of HSL⫺/⫺ and HSL⫹/⫹ mice to normalchow and high-fat diets, focusing mainly on the function of adipose tissues.

The costs of publication of this article were defrayed in part by thepayment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
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E1183RESISTANCE TO DIET-INDUCED OBESITY IN HSL-KO MICE

Table 1. Primers used for real-time RT-PCR

ACRP30
LeptinResistin
Adipsin
TNF␣
PPAR␥
PGC-1
C/EBP␣ChREBP
ADD1/SREBP-1c
SREBP-1a
SREBP-2
ALBP
Perilipin
LPL
GPAT
DGAT1
DGAT2
FAS
ACLY
INSR
IRS-1
GLUT4
HMGCS1
HMGCR
LDLR
ACAT1
UCP1
UCP2
28S rRNA

Forward

Reverse5⬘-CGGCAGCACTGGCAAGTT-3⬘
5⬘-AACCCTCATCAAGACCATTGTCA-3⬘
5⬘-GCTGCTGCCAAGGCTGAT-3⬘
5⬘-GCCTGATGTCCTGCATCAACT-3⬘
5⬘-CACAAGATGCTGGGACAGTGA-3⬘
5⬘-GCCCACCAACTTCGGAATC-3⬘
5⬘-GGACAGTCTCCCCGTGGAT-3⬘
5⬘-CGCAAGAGCCGAGATAAAGC-3⬘
5⬘-GGACAAGATCCGGCTGAACA-3⬘
5⬘-GGAGCCATGGATTGCACATT-3⬘
5⬘-ACAGCGGTTTTGAACGACATC-3⬘
5⬘-GCGCCAGGAGAACATGGT-3⬘
5⬘-GCGTGGAATTCGATGAAATCA-3⬘
5⬘-CATCTCTACCCGCCTTCGAA-3⬘
5⬘-AGTGGCCGAGAGCGAGAAC-3⬘
5⬘-CAACACCATCCCCGACATC-3⬘
5⬘-CATGCGTGATTATTGCATCCA-3⬘
5⬘-CCGCAAAGGCTTTGTGAAG-3⬘
5⬘-TCCTGGAACGAGAACACGATCT-3⬘
5⬘-TGGAGGCAGCATTGCAAAC-3⬘
5⬘-CGCTGTGTGAACTTCAGCTTCT-3⬘
5⬘-CCTCAGTCCCAACCATAACCA-3⬘
5⬘-CATGGCTGTCGCTGGTTTC-3⬘
5⬘-GACAAGAAGCCTGCTGCCATA-3⬘
5⬘-TGCACGGATCGTGAAGACA-3⬘
5⬘-GCTCCATAGGCTATCTGCTCTTCA-3⬘
5⬘-TGACTTTGTGACCAACCTCATTG-3⬘
5⬘-CGATGTCCATGTACACCAAGGA-3⬘
5⬘-GGCCTCTGGAAAGGGACTTC-3⬘
5⬘-GAATCCGCTAAGGAGTGTGTAACA-3⬘

5⬘-CCGTGATGTGGTAAGAGAAGTAGTAGA-3⬘
5⬘-CCTCTGCTTGGCGGATACC-3⬘
5⬘-TCTCCTTCCACCATGTAGTTT CC-3⬘
5⬘-GCGCAGATTGCAGGTTGTC-3⬘
5⬘-TCCTTGATGGTGGTGCATGA-3⬘
5⬘-TGCGAGTGGTCTTCCATCAC-3⬘
5⬘-TCCATCTGTCAGTGCATCAAATG-3⬘
5⬘-CGGTCATTGTCACTGGTCAACT-3⬘
5⬘-CGTCCGTTGCACATATTGAATG-3⬘
5⬘-GGAAGTCACTGTCTTGGTTGTTGA-3⬘
5⬘-GGAAGTCACTGTCTTGGTTGTTGA-3⬘
5⬘-CGATGCCCTTCAGGAGCTT-3⬘
5⬘-CCCGCCATCTAGGGTTATGA-3⬘
5⬘-TGCTTGCAATGGGCACACT-3⬘
5⬘-CCACCTCCGTGTAAATCAAGAAG-3⬘
5⬘-GTGACCTTCGATTATGCGATCA-3⬘
5⬘-GCCAGGCGCTTCTCAATCT-3⬘
5⬘-AGGAATAAGTGGGAACCAGATCAG-3⬘
5⬘-GAGACGTGTCACTCCTGGACTTG-3⬘
5⬘-TCTCACAATGCCCTTGAAGGT-3⬘
5⬘-CAGCCAGGCTTCCGAGAGT-3⬘
5⬘-CCGGCACCCTTGAGTGTCT-3⬘
5⬘-AAACCCATGCCGACAATGA-3⬘
5⬘-CGGCTTCACAAACCACAGTCT-3⬘
5⬘-GTCTCTCCATCAGTTTCTGAACCA-3⬘
5⬘-GCGGTCCAGGGTCATCTTC-3⬘
5⬘-GAGTGCACACCCACCATTGTC-3⬘
5⬘-TCGCAGAAAAGAAGCCACAA-3⬘
5⬘-ACCAGCTCAGCACAGTTGACA-3⬘
5⬘-CTCCAGCGCCATCCATTT-3⬘

ACRP30, adipocyte complement-related protein of 30 kDa; TNF-␣, tumor necrosis factor-␣; PPAR␥, peroxisome proliferator-activatedreceptor-␥; PGC-1, PPAR␥ coactivator 1; C/EBP␣, CAAT/enhancer-binding protein-␣; ChREBP, carbohydrate response element-binding
protein; ADD1, adipocyte determination- and differentiation-dependent factor 1; SREBP, sterol-regulatory element-binding protein; ALBP,
adipocyte lipid-binding protein; LPL, lipoprotein lipase; GPAT, glycerol-3-phosphate acyltransferase; DGAT, acyl-CoA:diacylglycerol acyltransferase; FAS, fatty acid synthase; ACLY, ATP citrate lyase; INSR, insulin receptor; IRS, insulin receptor substrate; GLUT, glucose
transporter; HMGCS, 3-hydroxy-3-methylglutaryl-CoA synthase; HMGCR, 3-hydroxy-3-methylglutaryl-CoA reductase; LDLR, low-density
lipoprotein receptor; ACAT1, acyl-CoA:cholesterol acyltransferase 1; UCP, uncoupling protein.

MATERIALS AND METHODS

Animals. HSL⫺/⫺ mice were generated by homologous
recombination as previously described (27). For breeding
experiments, mice heterozygous for the deleted HSL allele
were used to generate homozygous HSL⫺/⫺ mice andHSL⫹/⫹ wild-type littermates. Genotyping was performed bya single-step PCR using three primers, as described previously (27). All experiments reported here were performed
with 129/Sv-C57BL6 hybrid descendants. High-fat (35.9%
wt/wt lard fat) diet and control normal chow (4.8% wt/wt fat)diet were obtained from Research Diets (New Brunswick, NJ;
product nos. D12309 and D12310, respectively). Twelveweek-old male and female HSL⫹/⫹ and HSL⫺/⫺ littermate
mice were randomized to either high-fat or normal chow dietsad libitum for 15 wk. For the comparison of food intake,
HSL⫹/⫹ and HSL⫺/⫺ mice were housed individually, andfood consumption was measured. For assessment of fastinginduced weight loss, 24-wk-old mice were fasted from 5 PM
until 9 AM and body weights measured before and after the
fast. For all other conditions, mice were housed in groups of
one to four animals.
Temperature measurement. Body temperature was measured with a digital thermometer (model 421501; Extech
Instruments, Waltham, MA). Core and surface body temperatures were measured by placing the probe for 30 s in therectum and on the skin of the midabdominal region, respectively.

Fecal lipid analysis. Feces of 39-wk-old female mice thatwere fed high-fat diets and housed in metabolic cages werecollected for 48 h, weighed, and stored at ⫺80°C. Total
lipids in the collected feces and in the consumed diet wereextracted by the method of Folch et al. (6), and acylglycerol
content was determined using an enzymatic assay kit(Sigma, St. Louis, MO).
Tissue lipid analysis. Animals under anesthesia were
killed by exsanguination. Tissues were excised, weighed,quickly frozen, and stored at ⫺80°C. Total lipids wereextracted (6), and enzymatic assay kits (Sigma) were used
for the determination of triacylglycerol and total cholesterol.
Histological analysis. Tissues were fixed with neutralbuffered formalin and embedded in paraffin. Sections were
stained with hematoxylin and eosin. Samples were observed
under a Leica DM IRB microscope.
Blood chemistries. Blood samples were drawn by retroorbital puncture from animals in the fed or overnight (⬎16h)-fasted state, as indicated in the figure legends. Cardiac
puncture was performed at the time of death in the fed state.
Enzymatic assay kits were used for the determination ofserum glucose, triglyceride, total cholesterol (Sigma), andFFA (Wako, Richmond, VA). Serum insulin, adipocyte complement-related protein of 30 kDa (ACRP30)/adiponectin,
and leptin were measured using radioimmunoassay kits
(Linco Research, St. Charles, MO).

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E1184RESISTANCE TO DIET-INDUCED OBESITY IN HSL-KO MICE

Analysis of mRNA. Tissues were homogenized in TRIzol
reagent (Invitrogen, Carlsbad, CA), and total RNA was extracted. RNA samples were further purified using the
RNeasy kit (Qiagen, Valencia, CA) with RNase-free DNase I
treatment according to the manufacturer’s instructions. Total RNA (1 ␮g) was reverse-transcribed in a 20-␮l reaction
containing random primers and Superscript II enzyme (Invitrogen). Real-time PCR was performed with an ABI Prism
8500 system using SYBR Green Master Mix reagent (Applied
Biosystems, Foster City, CA) and specific primer pairs (Table
1) selected with Primer Express software (Applied Biosystems). The relative mass of specific RNAs was calculated by
the comparative cycle of threshold detection method according to the manufacturer’s instructions. Equal PCR efficiency
was ensured by control amplification using serial dilutions(1:1–1:128) of reverse-transcribed RNA. We confirmed theabsence of genomic DNA amplification or primer dimer formation by control amplifications using non-reverse-transcribed RNA or no addition of DNA as templates. Agarose gelelectrophoresis or dissociation curve analysis of RT-PCR
products was performed to ensure that a single amplicon was
obtained.

Statistical analysis. Results are given as means ⫾ SE, andstatistical significance was tested by unpaired two-tailed
Student’s t-test, except where otherwise stated, using StatView (version 4.5; Abacus Concepts, Berkeley, CA) and InStat (version 2.03; GraphPad Software, San Diego, CA) software for Macintosh. Data were analyzed separately for maleand female mice and the results combined unless sex differences were observed.
RESULTS

Resistance to high-fat diet-induced obesity in HSL⫺/⫺mice. Changes in body weights during 15 wk of conditioned feeding are shown in Fig. 1. Feeding of a normalchow diet resulted in similar small increments in bodyweight in both male and female mice, without any
differences between HSL⫺/⫺ and HSL⫹/⫹ mice. In
contrast, a high-fat diet was associated with a substantial weight gain in normal mice; however, the absenceof HSL resulted in a 20% lower (P ⬍ 0.01) body weightin male HSL⫺/⫺ mice than controls (Fig. 1A). The bodyFig. 1. Body weight, food intake andbody temperature of wild-type (HSL⫹/⫹,
closed symbols) and hormone-sensitive
lipase (HSL)-deficient (HSL⫺/⫺, open
symbols) mice. Twelve-week-old mice
were fed either normal chow (NC, 4.8%
wt/wt fat, circles) or high-fat (HF,
35.9% wt/wt fat, squares) diet for 15
wk. Body weight values of male (A) andfemale (B) mice. C: loss of body weightafter overnight fasting. Twenty-fourweek-old mice were fasted overnight,
and differences in body weights after
the fast are shown; n ⫽ 15–18. D: foodintake of HSL⫹/⫹ and HSL⫺/⫺ female
mice fed NC or HF diets (n ⫽ 8–30). E:
core and surface body temperature of
44-wk-old HSL⫹/⫹ and HSL⫺/⫺ female mice fed HF diets (n ⫽ 6). Results
are means ⫾ SE. *P ⬍ 0.05, **P ⬍ 0.01
compared with HSL⫹/⫹ mice undercorresponding condition.

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E1185RESISTANCE TO DIET-INDUCED OBESITY IN HSL-KO MICE

weight of female HSL⫺/⫺ mice was slightly higher
than that of HSL⫹/⫹ mice during the first 3 wk of ahigh-fat diet (Fig. 1B); however, after 10 wk of high-fat
feeding, body weight gain was markedly reduced infemale HSL⫺/⫺ mice compared with HSL⫹/⫹ mice.
Final body weight of high-fat-fed female HSL⫺/⫺ micewas 26% lower (P ⬍ 0.001) than that of HSL⫹/⫹ mice.
Because HSL has been reported to be expressed in
the small intestine and it has been suggested that HSL
might affect the absorption of lipids, especially of cholesteryl esters (7), fecal lipid content was measured toassess the potential malabsorption of dietary acylglycerols. No differences were found between HSL⫺/⫺ andcontrol mice in fecal appearance or weight (48 ⫾ 3 vs.
60 ⫾ 7 mg/g food intake; n ⫽ 4, P ⬎ 0.05). Furthermore,
fecal acylglycerol content was not different between
HSL⫺/⫺ and control mice (0.1 ⫾ 0.0 vs. 0.3 ⫾ 0.1% of
dietary acylglycerols; n ⫽ 4, P ⬎ 0.05), confirming thatboth HSL⫺/⫺ and control mice absorbed ⬎99.5% of
dietary acylglycerols. Thus malabsorption of acylglycerols is not responsible for the resistance to high-fat
diet-induced obesity in HSL⫺/⫺ mice. The amount offasting-induced weight loss, which provides a simple
approximation of energy expenditure because of theelimination of energy intake, is shown in Fig. 1C.
HSL⫺/⫺ mice lost ⬃1.5-fold more weight comparedwith controls. Consistent with the higher energy expenditure in HSL⫺/⫺ mice, food intake per body weight
was higher in HSL⫺/⫺ mice than in controls (Fig. 1D).
Core body temperatures were also higher in HSL⫺/⫺mice (Fig. 1E). We (27) previously reported no differences in oxygen consumption, respiratory quotient, or
core body temperature between HSL⫺/⫺ and wild-typemice on a normal chow diet, which is consistent withsimilar body weights of HSL⫺/⫺ and wild-type mice ona normal chow diet (Fig. 1, A and B). The present
results, however, suggest that, on a high-fat diet,
HSL⫺/⫺ mice display increased energy expenditurethrough elevated thermogenesis.
We next examined the weights of various internalorgans, including several different adipose tissue depots (Table 2). Even though total body weights weresimilar on a normal chow diet, total WAT mass, reflective of each of the fat depots (inguinal, femoral, scap-

ular, perigonadal, and retroperitoneal), was statistically reduced (P ⬍ 0.05) in HSL⫺/⫺ mice comparedwith controls. A high-fat diet resulted in large increases in WAT mass in all fat depots in normal mice;
however, there were few or no changes in WAT mass in
HSL⫺/⫺ mice. Thus, with a high-fat diet, all measured
WAT depots were significantly smaller in HSL⫺/⫺mice than in HSL⫹/⫹ mice, with total WAT weight⬎70% lower in HSL⫺/⫺ mice than in HSL⫹/⫹ mice
(P ⬍ 0.0001). The differences in total WAT weights
were 5.12 g in male and 8.24 g in female mice, which
contribute 56 and 68% of the lower body weights,
respectively. Differences in the percent weight of WAT
vs. body weight were still significant between HSL⫺/⫺and HSL⫹/⫹ mice although total body weights werelower in HSL⫺/⫺ mice on a high-fat diet (Fig. 2A). In
contrast to WAT, interscapular BAT was larger in
HSL⫺/⫺ mice than in controls (Table 2). This resulted
in BAT representing a greater percentage of total bodyweight in male and female HSL⫺/⫺ mice than in controls on either a normal chow or high-fat diet (Fig. 2B).
In view of the reduction in WAT weight in the presence of similar body weights on a normal chow diet,most of the internal organs, such as liver, spleen,pancreas, heart, and ovary (Table 2), had a tendency to
be larger in HSL⫺/⫺ mice than in HSL⫹/⫹ mice. This
was also true on a high-fat diet. For example, livers ofhigh-fat-fed HSL⫺/⫺ male mice were ⬃1.5-fold larger
than those in the corresponding controls (P ⬍ 0.05),
whereas spleens were 1.5- to 3.2-fold larger (P ⬍ 0.05
to P ⬍ 0.01) in high-fat-fed HSL⫺/⫺ mice than incontrols. One exception was the testis, which was⬃30% smaller in HSL⫺/⫺ mice than in HSL⫹/⫹ mice
under both normal chow and high-fat conditions, consistent with previous reports of severe oligo/azospermia in HSL⫺/⫺ mice (3, 27).
Lipid composition and histology of tissues. Although
WAT weight was lower in HSL⫺/⫺ mice and thesedifferences from controls were accentuated by high-fat
feeding, triacylglycerol content per gram of WAT
tended to be similar in female HSL⫺/⫺ and control
mice (Fig. 2C); however, triacylglycerol content of WAT
from high-fat-fed male HSL⫺/⫺ mice was 42% lower
than that of HSL⫹/⫹ mice (P ⬍ 0.01). Likewise, BAT,

Table 2. Weights of organs
Normal Chow

No. of mice and sex
Total WAT
Interscapular BAT
LiverSpleenPancreasHeart
Testis (male)
Ovary (female)High Fat

HSL⫹/⫹

HSL⫺/⫺

Foldincrease

HSL⫹/⫹

HSL⫺/⫺

Foldincrease

8M, 8F
3,131 ⫾ 389
404 ⫾ 53
1,417 ⫾ 101
99 ⫾ 6188 ⫾ 11147 ⫾ 6256 ⫾ 188⫾1

8M, 7F
1,980 ⫾ 170*673 ⫾ 66†
1,785 ⫾ 192
173 ⫾ 17†227 ⫾ 24
170 ⫾ 11154 ⫾ 7*20 ⫾ 5*

0.6
1.7
1.3
1.7
1.2
1.2
0.6
2.4

7M, 8F
9,287 ⫾ 919
837 ⫾ 98
1,900 ⫾ 158
112 ⫾ 7
195 ⫾ 18173 ⫾ 8249 ⫾ 1217 ⫾ 5

9M, 9F
2,478 ⫾ 213†
1,012 ⫾ 77
2,589 ⫾ 218*
274 ⫾ 42†237 ⫾ 14196 ⫾ 6*166 ⫾ 7*32 ⫾ 3*

0.3
1.2
1.4
2.4
1.2
1.1
0.7
1.9

Results are means ⫾ SE (in mg) of indicated no. of mice aged 27 wk in the fed state. WAT and BAT, white and brown adipose tissue,
respectively. Fold increase compares hormone-sensitive lipase (HSL)-deficient (HSL⫺/⫺) with wild-type (HSL⫹/⫹) mice. Total WAT mass isthe sum of inguinal, femoral, scapular, perigonadal, and retroperitoneal WAT depots. * P ⬍ 0.05, † P ⬍ 0.01 compared with HSL⫹/⫹ mice.
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E1186RESISTANCE TO DIET-INDUCED OBESITY IN HSL-KO MICE

Fig. 2. Percent weight of fat depots
and tissue lipid content. Percent organ
weights of total white (WAT; A) andinterscapular brown adipose tissue(BAT; B); n ⫽ 15–18 mice. Triacylglycerol (TG; C and D) and total cholesterol(TC; E and F) content in WAT (C andE) and BAT (D and F) of 27-wk-old fed
mice. Results are means ⫾ SE; n ⫽ 8
mice. *P ⬍ 0.05, **P ⬍ 0.01 comparedwith HSL⫹/⫹ mice under corresponding condition.

although tending to have greater weight in HSL⫺/⫺
mice, displayed similar triacylglycerol content pergram of tissue in HSL⫺/⫺ and control mice (Fig. 2D). In
contrast to triacylglycerol content, cholesterol content
in adipose tissues (WAT and BAT) tended to be higher
in HSL⫺/⫺ mice on a normal chow diet (Fig. 2, E and
F). High-fat feeding accentuated these differences, particularly in WAT, where cholesterol content was about
fivefold higher in male and female HSL⫺/⫺ mice (P ⬍0.01). These elevations in cholesterol content are compatible with the absence of neutral cholesteryl esterhydrolase (CEH) activity in adipose tissue (WAT and
BAT) of HSL⫺/⫺ mice (27).
Hepatic triacylglycerol content was higher in male
HSL⫺/⫺ mice than in controls (P ⬍ 0.05); however, nosignificant differences were observed in female mice
(Fig. 3A). High-fat feeding increased triacylglycerol
content further in both HSL⫺/⫺ and control male mice.
Hepatic cholesterol content was generally higher inmale and female HSL⫺/⫺ than in control mice whether

fed normal chow or high fat (Fig. 3B). Consistent withthe changes in lipid content, histological examinationof the liver revealed lipid accumulation in hepatocytesof male HSL⫺/⫺ mice (Fig. 3, C-F). On a normal chow
diet, hepatocyes of male HSL⫺/⫺ mice were larger andshowed lipid vacuolation (Fig. 3D), which resembles
that of high-fat-fed HSL⫹/⫹ control male mice (Fig.
3E). Lipid accumulation was even more pronounced inhigh-fat-fed HSL⫺/⫺ male mice (Fig. 3F). There wereno substantial morphological changes in female mice(data not shown), compatible with the analysis of thetriacylglycerol content.
Serum biochemical measurements. Serum biochemical values associated with lipid and carbohydrate metabolism are shown in Table 3. Fasting blood glucose
values tended to be lower in HSL⫺/⫺ mice, reachingstatistical significance, however, only in male mice fed
normal chow (40% decrease, P ⬍ 0.05). Fasting triglyceride and FFA levels were lower in HSL⫺/⫺ mice fed anormal chow diet, as previously reported (29, 35), and

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E1187RESISTANCE TO DIET-INDUCED OBESITY IN HSL-KO MICE

Fig. 3. Biochemical and histologicalchanges in livers. TG (A) and TC (B)content in livers of HSL⫺/⫺ andHSL⫹/⫹ mice. Total lipids were extracted from livers of 27-wk-old fed
male mice. Results are means ⫾ SE;n ⫽ 4 mice. *P ⬍ 0.05, **P ⬍ 0.01
compared with HSL⫹/⫹ mice undercorresponding condition. Hematoxylineosin stained liver samples from NCfed HSL⫹/⫹ (C), NC-fed HSL⫺/⫺ (D),HF-fed HSL⫹/⫹ (E), and HF-fed
HSL⫺/⫺ (F) mice. Original magnification, ⫻20. Scale bars, 50 ␮m.

these differences persisted after high-fat feeding. Totalcholesterol levels were not different between HSL⫺/⫺and control mice. The high-fat diet caused no significant changes in fasting glucose, triglyceride, FFA, orcholesterol concentrations in normal or HSL⫺/⫺ mice.

Fasting insulin concentrations were not significantly
different between control and HSL⫺/⫺ mice. In contrast, insulin values obtained in the fed state tended tobe higher in HSL⫺/⫺ mice, reaching statistical significance (P ⬍ 0.05) in normal chow-fed male animals;

Table 3. Serum biochemical values
Normal Chow

No. of mice and sex
Glucose, mg/dl
Triglyceride, mg/dl
Total cholesterol, mg/dl
FFA, mmol/l
No. of mice and sex
Insulin, fasted, ng/ml
No. of mice and sex
Insulin, fed, ng/ml

HSL⫹/⫹

HSL⫺/⫺

8M, 8F119 ⫾ 1282 ⫾ 6157 ⫾ 41.32 ⫾ 0.10
8M, 8F
0.68 ⫾ 0.10
4M, 5F2.06 ⫾ 0.36

8M, 7F88 ⫾ 1239 ⫾ 3†157 ⫾ 110.96 ⫾ 0.05†
8M, 6F
0.54 ⫾ 0.06
4M, 5F9.70 ⫾ 2.50

High Fat
Fold
increase

0.7
0.5
1.0
0.7
0.8
2.3

HSL⫹/⫹

HSL⫺/⫺

7M, 8F124 ⫾ 1483 ⫾ 6
182 ⫾ 101.08 ⫾ 0.06
7M, 8F1.24 ⫾ 0.29
3M, 4F7.43 ⫾ 2.28

9M, 9F92 ⫾ 8*50 ⫾ 8†183 ⫾ 80.80 ⫾ 0.07†
8M, 9F1.04 ⫾ 0.18
3M, 4F9.92 ⫾ 2.34

Foldincrease

0.7
0.6
1.0
0.7
0.8
1.3

Results are means ⫾ SE of indicated no. of mice. Fold increase compares HSL
with HSL
mice. Insulin values in the fed state wereobtained from 27-wk-old mice. Other samples were obtained from 24-wk-old mice after an overnight fast. * P ⬍ 0.05, † P ⬍ 0.01 compared with
⫹/⫹
HSL
mice.
⫺/⫺

⫹/⫹

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E1188RESISTANCE TO DIET-INDUCED OBESITY IN HSL-KO MICE

insulin increased similarly in both control and HSL⫺/⫺mice with the high-fat diet. There were no significant
differences in serum glucose, triglyceride, total cholesterol, or FFA levels between control and HSL⫺/⫺ micein the fed state (data not shown). Serum insulin values
in the fed state were positively correlated with liver
weights (Spearman r ⫽ 0.71, P ⬍ 0.0001).
Serum values of ACRP30/adiponectin (10, 15, 31)
and leptin (39), two adipocyte-derived hormones, were
measured to evaluate adipose tissue function. SerumACRP30/adiponectin levels were 75–90% lower (P ⬍0.01) in both male and female HSL⫺/⫺ mice comparedwith controls fed either normal chow or high-fat diets(Fig. 4A). When ACRP30/adiponectin values were plotted against body weights, HSL⫺/⫺ and control mice

distributed as two distinct clusters (Fig. 4B), with
log-transformed ACRP30 values linearly correlatedwith body weights in HSL⫺/⫺ mice (Pearson r ⫽ ⫺0.65,
P ⬍ 0.001). In contrast, ACRP30/adiponectin values ofboth HSL⫺/⫺ and HSL⫹/⫹ mice were distributed along
one regression line when plotted against either liver
weights (Fig. 4C; Pearson r ⫽ ⫺0.66, P ⬍ 0.0001) or
hepatic triacylglycerol contents (Pearson r ⫽ ⫺0.61,
P ⬍ 0.001). Serum ACRP30/adiponectin values were
negatively correlated with insulin values in the fed
state (Spearman r ⫽ ⫺0.52, P ⬍ 0.01).
Circulating serum leptin values were not significantly different between control and HSL⫺/⫺ mice fed
normal chow (Fig. 4D). Serum leptin values increased
with the high-fat diet in control mice; however, noFig. 4. Serum values of adipocyte complement-related protein of 30 kDa(ACRP30) and leptin. Serum ACRP30/
adiponectin (A) and leptin (B) from 24wk-old mice (n ⫽ 6) after overnight fast
and from 27-wk-old fed mice (n ⫽ 7–9),
respectively. Results are means ⫾ SE.
*P ⬍ 0.05, **P ⬍ 0.01 compared with
HSL⫹/⫹ mice under corresponding
condition. Serum values of ACRP30 (Cand E) and leptin (D and F) are plottedagainst body weights (C and D) andliver weights (E and F).

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RESISTANCE TO DIET-INDUCED OBESITY IN HSL-KO MICE

changes were observed in HSL⫺/⫺ mice, resulting inserum leptin concentrations that were 66 and 82%
lower in male and female HSL⫺/⫺ mice on a high-fat
diet (P ⬍ 0.01 and ⬍ 0.0001, respectively). Log-transformed leptin values of control mice correlated linearlywith body weights (Fig. 4E; Pearson r ⫽ 0.83, P ⬍0.0001). Leptin values observed in HSL⫺/⫺ mice witheither normal chow or high-fat feeding were significantly lower than the values predicted by this correlation (paired t-test, P ⬍ 0.01 and ⬍ 0.001, respectively).
Therefore, the decrease in leptin values in HSL⫺/⫺mice was more than could be explained by the decreasein body weights. In contrast to ACRP30, no significant
correlation was observed between serum leptin values
and either liver weights (Fig. 4F; Pearson r ⫽ 0.14, P ⫽0.27) or hepatic triacylglycerol contents (Pearson r ⫽⫺0.13, P ⫽ 0.46). Serum insulin values in the fed state
were not correlated with serum leptin values (Spearman r ⫽ 0.20, P ⫽ 0.12).

E1189

Expression of genes in WAT and BAT of HSL⫺/⫺mice. In view of the lower circulating concentrations of
ACRP30 and leptin in HSL⫺/⫺ mice, expression levelsof ACRP30/adiponectin (Fig. 5A) and leptin (Fig. 5B)
mRNA in WAT were assessed by real-time RT-PCR.
Consistent with the circulating values, both ACRP30/
adiponectin and leptin mRNA levels were ⬃75% lower
in HSL⫺/⫺ mice on normal chow or high-fat diets.
Significant correlation was observed between circulating values and mRNA levels for both ACRP30/adiponectin (Spearman r ⫽ 0.85, P ⬍ 0.0001) and leptin(Spearman r ⫽ 0.82, P ⬍ 0.0001), suggesting that
decreased production of these hormones in HSL⫺/⫺mice contributed to the lower circulating values. In
contrast to WAT, expression of ACRP30/adiponectin in
BAT was not different between HSL⫺/⫺ and control
mice (data not shown). Expression of leptin in BAT of
HSL⫺/⫺ mice was lower than in controls only withhigh-fat feeding (data not shown).

Fig. 5. Expression of adipose-derived
humoral factors. Total RNA extracted
from perigonadal WAT of 27-wk-old fed
mice was used for gene expression
analysis of ACRP30 (A), leptin (B), resistin (C), adipsin (D), and TNF-␣ (E)by real-time RT-PCR. Levels of 28S
rRNA were used for normalization ofsample loading. Level of mRNA expression observed in wild-type mice on
the NC diet was set as 100% control.
Results are means ⫾ SE; n ⫽ 4 maleand 4 female mice per group. *P ⬍0.05, **P ⬍ 0.01 compared with
HSL⫹/⫹ mice under corresponding
condition.

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E1190RESISTANCE TO DIET-INDUCED OBESITY IN HSL-KO MICE

We next examined the expression of other humoral
factors derived from adipose tissues (21). Expression ofresistin and adipsin was decreased 60–90% (P ⬍ 0.01)
both in WAT (Fig. 5, C and D) and BAT (data notshown) of HSL⫺/⫺ mice. In contrast, expression of
TNF-␣ was upregulated 2- to 3-fold (P ⬍ 0.01) in WAT(Fig. 5E) and 5- to 10-fold (P ⬍ 0.01) in BAT (data notshown) of HSL⫺/⫺ mice compared with control.
To further explore the effects of HSL deficiency on
adipose metabolism, we examined the expression oftranscription factors associated with adipogenesis.
Levels of expression of peroxisome proliferator-activated receptor-␥ (PPAR␥) and CCAAT/enhancer-binding protein-␣ (C/EBP␣), two of the major transcription
factors for adipogenesis (30, 36), were suppressed 40–
70% (P ⬍ 0.01) in WAT (Fig. 6, A and B) of both maleand female HSL⫺/⫺ compared with control mice, although differences were more prominent in male mice(data not shown). Expression levels of lipogenic tran-

scription factors (26, 32, 33) such as carbohydrate response element-binding protein (ChREBP) and adipocyte determination- and differentiation-dependent factor 1/sterol-regulatory element-binding protein-1c(ADD1/SREBP-1c) were also suppressed 50–75% (P ⬍0.05) in WAT of HSL⫺/⫺ mice (Fig. 6, C and D).
Expression of SREBP-1a, which controls both triglyceride synthesis and cholesterol metabolism, was notaffected by HSL deficiency in WAT (data not shown).
However, SREBP-2, which controls cholesterol synthesis and uptake, was upregulated twofold (P ⬍ 0.05) in
WAT of HSL⫺/⫺ mice (Fig. 6E). In contrast to WAT,
expression levels of PPAR␥, C/EBP␣, ChREBP, and
ADD1/SREBP-1c, as well as expression of PPAR␥ coactivator-1 (PGC-1), were unaffected by the absence of
HSL in BAT (data not shown). However, expression
levels of SREBP-1a and SREBP-2 in BAT were increased two- to threefold (P ⬍ 0.01) in HSL⫺/⫺ mice(data not shown).

Fig. 6. Expression of transcription factors associated with adipogenesis andlipid metabolism. Total RNA extracted
from perigonadal WAT of 27-wk-old fed
mice was used for mRNA expression
analysis of peroxisome proliferator-activated receptor-␥ (PPAR␥; A), CAAT/
enhancer-binding protein-␣ (C/EBP␣;B), carbohydrate response elementbinding protein (ChREBP; C), adipocyte determination- and differentiation-dependent factor 1/sterol-regulatory element-binding protein-1 (ADD1/SREBP-1c; D), and SREBP-2 (E) by
real-time RT-PCR, as described in Fig.
5. Level of mRNA expression observedin wild-type mice on the NC diet was
set as 100% control. Results aremeans ⫾ SE; n ⫽ 4 male and 4 female
mice per group. *P ⬍ 0.05, **P ⬍ 0.01
compared with HSL⫹/⫹ mice undercorresponding condition.

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RESISTANCE TO DIET-INDUCED OBESITY IN HSL-KO MICE

In parallel with the suppression of adipogenic transcription factors PPAR␥ and C/EBP␣ in WAT of
HSL⫺/⫺ mice, expression of adipose differentiation
markers, such as ALBP and perilipin, was suppressed
50–80% (P ⬍ 0.01; Fig. 7, A and B). Expression of
lipoprotein lipase (LPL) was also suppressed 30–40%(P ⬍ 0.05) in WAT of HSL⫺/⫺ mice (Fig. 7C), withgreater reductions observed in male mice. The levels of
expression of enzymes for triglyceride synthesis, suchas glycerol-3-phosphate acyltransferase (GPAT; Fig.
7D) and fatty acid synthase (FAS; Fig. 7E), as well as
acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1),
DGAT2, and ATP citrate lyase (ACLY) (data notshown), were decreased 60–80% (P ⬍ 0.01) in WAT of
HSL⫺/⫺ mice. Expressions of these adipocyte markers
and lipogenic enzymes in BAT were not affected by theabsence of HSL (data not shown), in parallel with thelevels of PPAR␥ and C/EBP␣ mRNA. LPL mRNAlevels in BAT were 1.7-fold higher in male HSL⫺/⫺

E1191

mice fed a normal chow diet compared with controls(P ⬍ 0.05).
Next, we examined the gene expression of some steps
in insulin-signaling pathways, since differentiationfrom preadipocytes to mature adipocytes is known to
be associated with an increase in insulin sensitivity(36). In accord with the downregulation of adipogenictranscription factors, expression of insulin receptor(INSR; Fig. 8A), insulin receptor substrate-1 (IRS-1;
Fig. 8B), and glucose transporter 4 (GLUT4) (data notshown) mRNA was decreased 30–80% (P ⬍ 0.01) in
WAT of HSL⫺/⫺ mice compared with controls. The
expression of these genes was not affected in BAT of
HSL⫺/⫺ mice (data not shown).
Because HSL appears to be the only enzyme that has
neutral CEH activity in both WAT and BAT (27), theexpression of genes involved with cholesterol metabolism was also examined. Expression of 3-hydroxy-3methylglutaryl-CoA reductase (HMGCR), the rate-lim-

Fig. 7. Expression of genes associatedwith triglyceride metabolism. Total
RNA extracted from perigonadal WATof 27-wk-old fed mice was used formRNA expression analysis of adipocyte lipid-binding protein (ALBP; A),perilipin (B), lipoprotein lipase (LPL;
C), glycerol-3-phosphate acyltransferase (GPAT; D), and fatty acid synthase (FAS; E) by real-time RT-PCR,
as described in Fig. 5. Level of mRNA
expression observed in wild-type miceon the NC diet was set as 100% control.
Results are means ⫾ SE; n ⫽ 4 maleand 4 female mice per group. *P ⬍0.05, **P ⬍ 0.01 compared with
HSL⫹/⫹ mice under corresponding
condition.

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E1192RESISTANCE TO DIET-INDUCED OBESITY IN HSL-KO MICE

Fig. 8. Expression of genes associatedwith insulin signaling, cholesterol metabolism, and thermogenesis. Total
RNA extracted from perigonadal WAT(A-E) and interscapular BAT (F) of 27wk-old fed mice was used for mRNA
expression analysis of insulin receptor(INSR; A), insulin receptor substrate-1(IRS-1; B), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR; C),
3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1; D), acyl-CoA:cholesterol acyltransferase 1 (ACAT1; E),
and uncoupling protein-2 (UCP2; F) by
real-time RT-PCR, as described in Fig.
5. Level of mRNA expression observedin wild-type mice on the NC diet was
set as 100% control. Results aremeans ⫾ SE; n ⫽ 4 male and 4 female
mice per group. *P ⬍ 0.05, **P ⬍ 0.01
compared with HSL⫹/⫹ mice undercorresponding condition.

iting enzyme for cholesterol synthesis, was upregulated about twofold (P ⬍ 0.05) in WAT and BATof HSL⫺/⫺ mice in parallel with SREBP-2 (Fig. 8C).
However, 3-hydroxy-3-methylglutaryl-CoA synthase-1(HMGCS1), an enzyme upstream of HMGCR, wasdownregulated 50–70% (P ⬍ 0.01) in WAT, but unchanged in BAT (data not shown), of HSL⫺/⫺ mice
(Fig. 8D); expression of low-density lipoprotein receptor (LDLR) mRNA was not affected by HSL deficiency
in either WAT or BAT (data not shown). Surprisingly,
expression of acyl-CoA:cholesterol acyltransferase 1(ACAT1), the enzyme that mediates the esterificationof cholesterol to cellular cholesteryl esters, was increased two- to fourfold (P ⬍ 0.01) in WAT and five- toeightfold (P ⬍ 0.01) in BAT (data not shown) of
HSL⫺/⫺ mice (Fig. 8E).
Finally, we examined the expression of uncoupling
proteins (UCP), since it appears that HSL⫺/⫺ mice
expend more energy in thermogenesis than control

mice. UCP2 is known to be expressed in several organs
including WAT and BAT, whereas UCP1 is expressed
specifically in BAT. Expression of UCP2 in WAT was
not altered by the absence of HSL in male mice but wasincreased in female HSL⫺/⫺ mice (data not shown).
However, expression of UCP2 in BAT was increased
three- to fourfold (P ⬍ 0.01) in both male and female
HSL⫺/⫺ mice (Fig. 8F), whereas expression of UCP1
was not changed (data not shown). The observed
amount of fasting-induced loss of body weight correlated with UCP2 expression (Pearson r ⫽ 0.65, P ⬍0.0001) but not with UCP1 expression (Pearson r ⫽0.01, P ⫽ 0.97) in BAT.
DISCUSSION

HSL⫺/⫺ mice have a substantial defect or complete
absence of glycerol release from isolated adipose cells
when stimulated by catecholamines; although FFA re-

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RESISTANCE TO DIET-INDUCED OBESITY IN HSL-KO MICE

lease occurs, it is significantly attenuated (8, 27, 35).
This defective release of glycerol and FFA from adipose
cells is accompanied by a marked accumulation of
diacylglycerol (8). Thus HSL appears to be the ratelimiting enzyme for diacylglycerol hydrolysis and to be
essential for hormone-stimulated lipolysis. Even
though lipolysis and the ability to release stored triacylglycerol from adipose tissue are dramatically reducedin HSL⫺/⫺ mice, these animals are not obese on anormal chow diet, although their adipose cells tend tobe hypertrophic and to display size heterogeneity.
In the present work, we have examined whether
feeding a high-fat diet would bring out a more apparent
phenotype in HSL⫺/⫺ mice and have explored specificchanges in gene expression. Even though HSL⫺/⫺ mice
have body weights similar to controls on a normal chow
diet, we found the total amount of WAT to be reduced.
Moreover, the amount of body weight gain produced
during high-fat feeding, i.e., dietary fat-induced obesity, was markedly reduced in both male and female
HSL⫺/⫺ mice compared with controls. Thus, although
the amount of WAT mass increased substantially incontrols fed a high-fat diet, there were minimalchanges in HSL⫺/⫺ mice, resulting in markedly lower
amounts of WAT in high-fat-fed HSL⫺/⫺ mice compared with controls. This resistance to weight gain andadiposity was not due to differences in food intake,since HSL⫺/⫺ mice had higher food consumption thancontrols when it was expressed as a function of bodyweight. In contrast to the smaller WAT depots in
HSL⫺/⫺ mice, BAT depots were increased in HSL⫺/⫺mice. The increase in BAT in HSL⫺/⫺ mice was accompanied by higher core body temperatures and an exaggerated weight loss induced by fasting. Therefore, resistance to high-fat diet-induced obesity in HSL⫺/⫺mice appears to be due to increased energy expenditureand thermogenesis in BAT. Consistent with this conclusion, the expression of UCP2 mRNA was increasedin BAT of HSL⫺/⫺ mice. Indeed, the observed amountof fasting-induced body weight loss in mice correlatedwith UCP2, but not with UCP1, expression in BAT.
These observations are in contrast to the effects of HSL
deficiency in the background of leptin deficiency(Sekiya M, Osuga J, and Ishihashi S, unpublishedobservations). Whereas the introduction of HSL deficiency into a genetic background of leptin deficiency
also results in lower body weights and adiposity, alower food intake without any effects on energy expenditure appears to be the mechanism responsible in thissetting. Thus leptin appears to modulate the mechanistic energy response (consumption vs. utilization) to
HSL deficiency.
Although WAT was reduced in HSL⫺/⫺ mice, WAT
was present in all normal depots, and there was no
obvious evidence of lipodystrophy. Nonetheless, thelevels of expression of transcription factors required for
adipocyte differentiation (PPAR␥, C/EBP␣), markers of
adipocyte differentiation (ACRP30/adiponectin, leptin,
resistin, adipsin, ALBP, perilipin, and LPL), and enzymes involved in triglyceride synthesis (GPAT,
DGAT1, DGAT2, FAS, and ACLY) were reduced in

E1193

WAT of HSL⫺/⫺ mice. HSL is presumably not required
for adipose differentiation, as embryonic fibroblasts
from HSL⫺/⫺ mice are able to be differentiated into
mature adipose cells in vitro (25). However, it is possible that HSL, although not required for initiation or
early stages of adipocyte differentiation, is needed under in vivo conditions for terminal differentiation. Thissuggestion is further supported by the finding of increased numbers of preadipocytes in the WAT depots ofLepob/obHSL⫺/⫺ mice (Sekiya M, Osuga J, and Ishihashi S, unpublished observations). Adipocyte differentiation is a complex process involving a cascade ofmany transcription factors, such as PPAR␥ and
C/EBP␣, and the expression of many adipocyte-specificgenes (4, 17). PPAR␥, a member of the nuclear hormone receptor family, is known to bind a variety offatty acids and fatty acid metabolites (1, 24). Because
HSL mediates the mobilization of fatty acids by hydrolysis of tri-, di-, and monoacylglycerols, as well as
cholesteryl esters, it might play an important role insupplying intrinsic ligands for PPAR␥. A relative lackof PPAR␥ ligands due to the absence of HSL might
suppress the mutual activation of PPAR␥ and C/EBP␣,thus affecting adipocyte differentiation in vivo. In thiscircumstance, the normal differentiation observed ofembryonic fibroblasts from HSL⫺/⫺ mice might be dueto the addition of exogenous PPAR␥ ligands to the cell
culture, thereby bypassing the requirement for endogenous PPAR␥ ligands to be produced by HSL-mediatedhydrolysis. Conversely, it is possible that the accumulation of diacylglycerol in WAT of HSL⫺/⫺ mice interferes with any number of orchestrated events involvedin normal differentiation rather than the deficiency ofan intrinsic ligand. It is important to note that theexpression of not all examined genes was reduced in
WAT from HSL⫺/⫺ mice. For instance, TNF-␣, ACAT,HMGCR, and SREBP-2 mRNA levels were all increased in WAT from HSL⫺/⫺ mice. The elevations in
ACAT, HMGCR, and SREBP-2 mRNA levels are interesting in light of the marked increase in cholesterol
content of WAT observed in HSL⫺/⫺ mice that was
accentuated by high-fat feeding. It is likely that theabsence of neutral CEH activity in WAT of HSL⫺/⫺mice causes a reduction in regulatory pools of cellular
unesterified cholesterol, leading to upregulation of
SREBP-2 and, subsequently, other sterol-regulated
genes (2). However, this cannot be the complete explanation, since LDLR mRNA levels are unchanged and
HMGCS mRNA levels are actually decreased.
In contrast to WAT, gene expression in BAT was
generally unaltered by the absence of HSL, eventhough BAT depots were increased. Nonetheless, similar to WAT, the ACAT, HMGCR, and SREBP-2 mRNAlevels were all elevated in BAT from HSL⫺/⫺ mice,
which occurred concurrently with an increase in cholesterol content, particularly with high-fat feeding.
Levels of UCP2 mRNA were elevated in BAT of bothmale and female HSL⫺/⫺ mice. The upregulation of
UCP2 paralleled the increase in SREBP-2 expressionin BAT, which is consistent with the activation of the
UCP2 promoter by SREBPs (19). Interestingly, UCP1

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E1194RESISTANCE TO DIET-INDUCED OBESITY IN HSL-KO MICEmRNA levels were unaltered, as were levels of PGC-1,
which has been linked to mitochondrial biogenesis andthermogenesis (28). Thus it is possible that the increased energy expenditure observed in HSL⫺/⫺ miceis due to the absence of neutral CEH activity in BAT
causing a reduction in regulatory pools of cellular unesterified cholesterol, leading to upregulation of
SREBP-2 and subsequent upregulation of UCP2 in
BAT, resulting in an increase in thermogenesis.
Liver weight was increased in HSL⫺/⫺ mice. Hepatic
content of both triacylglycerol and cholesterol of
HSL⫺/⫺ mice was about twice that of controls. Histological features of the liver clearly supported the accumulation of lipid in hepatocytes of HSL⫺/⫺ male mice.
Because the liver is not an organ where HSL is normally expressed, our findings suggest that HSL deficiency affects hepatic lipid metabolism indirectly. On
the basis of the correlation of liver weight with both
serum ACRP30/adiponectin and insulin levels, but notwith leptin, we speculate that decreased ACRP30/adiponectin levels in HSL⫺/⫺ mice play a causative role inhepatic fat accumulation, possibly through inactivation of fatty acid oxidation (37), and induction of fatty
acid synthesis by hyperinsulinemia in the fed state; theexpression of lipogenic enzymes such as FAS, GPAT,
and ACLY are increased in the liver of HSL⫺/⫺ mice(Harada K, Shen W-J, and Kraemer FB, unpublishedobservations). In contrast to these observations, otherinvestigators (8, 9, 35) have reported that hepatic triacylglycerol content of HSL⫺/⫺ mice was lower than incontrols. The basis for the difference is that these otherinvestigators showed that fasting induced an increaseof hepatic triacylglycerol in control mice, but not in
HSL⫺/⫺ mice, where circulating FFA levels failed torise to the levels seen in controls.
Fasting blood glucose, triglyceride, and FFA levels
were lower in HSL⫺/⫺ mice than in controls on either
a normal chow or high-fat diet. There was no differencein fasting insulin level between HSL⫺/⫺ and control
mice, consistent with a previous report (29), and fasting insulin values rose on a high-fat diet similarly incontrol and HSL⫺/⫺ mice. However, insulin levels in
the fed state were higher in HSL⫺/⫺ male mice than incontrols. This might indicate the existence of insulin
resistance in HSL⫺/⫺ mice, even though they have lessadiposity. This would be compatible with the decreased
expression of INSR, IRS-1, and GLUT4, accompaniedby the decrease of C/EBP␣ (36), observed in WAT of
HSL⫺/⫺ mice. Lower circulating ACRP30/adiponectinlevels in HSL⫺/⫺ mice might contribute to this, since
deficiency of ACRP30/adiponectin has been reported tocause insulin resistance and hyperinsulinemia (13, 16).
Another possible mechanism contributing to the potential insulin resistance in HSL⫺/⫺ mice is the apparent
upregulation of adipose tissue TNF-␣, a cytokine associated with inflammation and known to cause insulin
resistance (34). It is possible that the reduced expression of PPAR␥, which has been shown to have ananti-inflammatory effect (5), in WAT of HSL⫺/⫺ mice
might allow the upregulation of TNF-␣. Interestingly,
we found that the size of pancreatic islets of HSL⫺/⫺mice was larger than that of controls (Shen W-J and
Kraemer FB, unpublished observations). This enlargement of islets could be a compensatory response toperipheral insulin resistance or could result from abnormalities in insulin secretion due to the absence of␤-cell HSL (29). Consistent with our findings, a recent
study reported that a different HSL⫺/⫺ mouse with ahybrid background displayed impaired insulin sensitivity in vivo associated with compensatory hypertrophy of pancreatic islets (23). It is noteworthy that
glucose-stimulated insulin secretion has been reported
to be intact in one strain of HSL⫺/⫺ mice (23) but
impaired in another HSL⫺/⫺ mouse with a diabetesprone C57BL/6 background (29).
In conclusion, HSL⫺/⫺ mice have reduced quantities
of WAT with increased amounts of BAT and are resistant to high-fat diet-induced obesity secondary to an
apparent increase in thermogenesis and energy expenditure. Although the accumulation of diacylglycerol intissues of HSL-deficient mice could be responsible forthese effects, the changes in adipose tissue gene expression in HSL⫺/⫺ mice suggest that abnormalities in
the generation of fatty acids or fatty acid metabolites
might prevent complete differentiation of WAT, resulting in alterations in adipocyte-derived hormones andcytokines with systemic manifestations. In addition, it
is suggested that dysregulation of intracellular cholesterol metabolism due to the inability to hydrolyze cholesteryl esters in adipose tissue of HSL-deficient mice
might be the primary abnormality leading to increased
thermogenesis. Thus the present experiments establish a potential link between adipose tissue sterol metabolism and energy expenditure. Additional experiments will be needed to prove this.
We thank Shahrad Taheri for suggestions and technical support,
Ann Nomoto for microscopic technique, and Eve Reaven, Salman
Azhar, Jingwen Liu, Penelope Collins, Yu Liang, and Jenny Wang
for helpful discussions.
DISCLOSURESThis work was supported, in part, by research grants from the
Research Service of the Department of Veterans Affairs, by Grant
DK-46942 from the National Institute of Diabetes and Digestive andKidney Diseases (to F. B. Kraemer), and by a Research Award fromthe American Diabetes Association (to W.-J. Shen).
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