THE JOURNAL OF BIOLOGICAL CHEMISTRY© 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 277, No. 22, Issue of May 31, pp. 19353–19357, 2002
Printed in U.S.A.

Absence of Sterol Regulatory Element-binding Protein-1 (SREBP-1)
Ameliorates Fatty Livers but Not Obesity or Insulin Resistance in
Lepob/Lepob Mice*
Received for publication, February 15, 2002, and in revised form, March 26, 2002
Published, JBC Papers in Press, March 28, 2002, DOI 10.1074/jbc.M201584200

Naoya Yahagi‡, Hitoshi Shimano§¶, Alyssa H. Hasty‡储, Takashi Matsuzaka§, Tomohiro Ide§,
Tomohiro Yoshikawa‡, Michiyo Amemiya-Kudo‡, Sachiko Tomita‡, Hiroaki Okazaki‡,
Yoshiaki Tamura‡, Yoko Iizuka‡, Ken Ohashi‡, Jun-ichi Osuga‡, Kenji Harada‡,
Takanari Gotoda‡, Ryozo Nagai‡, Shun Ishibashi‡, and Nobuhiro Yamada§From the ‡Department of Internal Medicine, University of Tokyo Graduate School of Medicine, Tokyo 113-8655 and§Department of Internal Medicine, Institute of Clinical Medicine, University of Tsukuba, Ibaraki 305-8575, Japan

Obesity is a common nutritional problem often associated with diabetes, insulin resistance, and fatty liver(excess fat deposition in liver). Leptin-deficient Lepob/
Lepob mice develop obesity and those obesity-related
syndromes. Increased lipogenesis in both liver and adipose tissue of these mice has been suggested. We havepreviously shown that the transcription factor sterol
regulatory element-binding protein-1 (SREBP-1) plays acrucial role in the regulation of lipogenesis in vivo. To
explore the possible involvement of SREBP-1 in thepathogenesis of obesity and its related syndromes, we
generated mice deficient in both leptin and SREBP-1. Indoubly mutant Lepob/ob ⴛ Srebp-1ⴚ/ⴚ mice, fatty livers
were markedly attenuated, but obesity and insulin resistance remained persistent. The mRNA levels of lipogenic enzymes such as fatty acid synthase were proportional to triglyceride accumulation in liver. In contrast,
the mRNA abundance of SREBP-1 and lipogenic enzymes in the adipose tissue of Lepob/Lepob mice was profoundly decreased despite sustained fat, which could
explain why the SREBP-1 disruption had little effect on
obesity. In conclusion, SREBP-1 regulation of lipogenesis is highly involved in the development of fatty livers
but does not seem to be a determinant of obesity in
Lepob/Lepob mice.

Obesity is the most common nutritional problem in the
United States, affecting ⬃33% of adults (1), and is often associated with type 2 diabetes due to insulin resistance (2).
The genetically obese Lepob/Lepob (Lepob/ob) mice develop obesity, glucose intolerance, insulin resistance, and fatty livers(excess fat deposition in liver) due to an inherited deficiency of
the appetite-suppressing hormone, leptin (3–7). They present
the most severe obesity ever known in both rodents and humans (8), and provide a good model of obesity and its related
syndromes including insulin resistance and fatty liver disease.

It has been reported that lipogenesis in both liver and adipose tissue is greater in obese animals than in lean controls (5,6). The livers of Lepob/ob mice have an increase in triglyceride
content, probably due to the increased lipogenesis paralleled byelevated mRNA expression and enzymatic activity of several
lipogenic enzymes such as fatty acid synthase and ATP citratelyase (5, 6, 9).
Sterol regulatory element-binding proteins (SREBPs)1 aretranscription factors that belong to the basic helix-loop-helixleucine zipper family and regulate enzymes responsible for thesynthesis of cholesterol, fatty acids, and triglycerides (10, 11).
To date, three SREBP isoforms, SREBP-1a, -1c, and -2, havebeen identified and characterized. SREBP-1a and -1c are transcribed from the same gene, each by a distinct promoter, andthe predominant SREBP-1 isoform in liver and adipose tissue
is SREBP-1c. Whereas SREBP-2 is relatively selective in transcriptionally activating cholesterol biosynthetic genes,
SREBP-1c has a greater role in regulating fatty acid synthesisthan cholesterol synthesis (12–15). SREBP-1c was also identified as adipocyte determination and differentiation factor-1
expressed in adipocytes and regulated during determinationand differentiation of cultured adipocyte cell lines (16). Thus,
the role of SREBP-1 in the regulation of lipogenesis has been
established (15, 17–19). Moreover, SREBP-1 now appears to bepositioned as a general mediator in the transcriptional action of
insulin (20 –22).
Recently, it has been reported that both SREBP-1c mRNAand its active nuclear protein are increased in Lepob/ob mouse
livers (23). It is pathophysiologically intriguing and of clinicalrelevance to evaluate the possible involvement of SREBP-1 inthe development of obesity and its related syndromes, since
SREBP-1 could be a potential therapeutic target in these pathological states. These considerations prompted us to investigate the effects of SREBP-1 deletion on the phenotype of
Lepob/ob mouse by targeted gene disruption.
EXPERIMENTAL PROCEDURES* This study was supported by the Promotion of Fundamental Studies in Health Science of the Organization for Pharmaceutical Safety andResearch. 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: Dept. of Internal
Medicine, Institute of Clinical Medicine, University of Tsukuba, 1-1-1
Tennodai, Tsukuba, Ibaraki 305-8575, Japan. Tel./Fax: 81-298-63-2170;
E-mail: shimano-tky@umin.ac.jp.
储 Present address: Division of Cardiovascular Medicine, Vanderbilt
University School of Medicine, 383 Preston Research Building, Nashville, TN 37232-6300
This paper is available on line at http://www.jbc.org

Animals—Mice deficient in SREBP-1 prepared as previously described (24) were back-crossed six times into the C57BL/6J backgroundand intercrossed with animals heterozygous at the leptin locus (Lep⫹/ob
C57BL/6J; Jackson Laboratories, Bar Harbor, ME) to generate double
heterozygotes. These mice were then interbred to produce Lepob/ob mice
whose Srebp-1 genotypes were either wild-type (Lepob/ob), heterozygous(Lepob/ob ⫻ Srebp-1⫹/⫺), or homozygous (Lepob/ob ⫻ Srebp-1⫺/⫺). The
frequency for obtaining the Lepob/ob ⫻ Srebp-1⫺/⫺ progeny was as low as

1
The abbreviations used are: SREBP, sterol regulatory elementbinding protein; PPAR␥, peroxisome proliferator-activated receptor ␥;
TNF␣, tumor necrosis factor ␣.

19353

SREBP-1 Disruption in Lepob/Lepob Mice

19354

TABLE I
Phenotypic characteristics of wild-type, Lepob/ob, and Lepob/ob ⫻ Srebp-1⫺/⫺ mice
Each value represents the mean ⫾ S.E. Mice used were 12 weeks of age, fed for 12 h after 24-h starvation. *, significance versus Lepob/ob at p ⬍0.05 by Student’s t test. Other parameters listed above have no significant difference between Lepob/ob and Lepob/ob ⫻ Srebp-1⫺/⫺ genotypes. NEFA,
nonesterified fatty acid.
Genotype

Wild type

Lepob/ob

Lepob/ob ⫻ Srebp-1⫺/⫺

Sex
Body weight (g)Epididymal fat weight (g)Liver cholesterol (mg/g)
Plasma cholesterol (mg/dl)
Plasma triglyceride (mg/dl)
Plasma NEFA (␮mol/l)
Plasma glucose (mg/dl)
Plasma insulin (ng/ml)

6 males27.1 ⫾ 0.25
0.16 ⫾ 0.03
2.8 ⫾ 0.31
63.0 ⫾ 7.8
84 ⫾ 9.1
192 ⫾ 70160 ⫾ 102.0 ⫾ 0.41

6 males, 6 females
43.3 ⫾ 2.4
2.7 ⫾ 0.32
3.2 ⫾ 1.1
118 ⫾ 1195 ⫾ 6.4
543 ⫾ 53278 ⫾ 19162 ⫾ 25

6 males, 6 females
44.8 ⫾ 1.3
3.1 ⫾ 0.28
4.5 ⫾ 0.84
89 ⫾ 4.1*
106 ⫾ 14427 ⫾ 55303 ⫾ 27141 ⫾ 31

1 in 43 of all pups born, probably due to the partial embryonic lethalityof SREBP-1-null mice (24). Genotypes at the SREBP-1 locus were
determined by Southern blot analysis with BamHI digestion (24). Genotypes at the leptin locus were determined by a PCR-based restrictedfragment length polymorphism analysis; DNA fragments amplified
from genomic DNA by PCR using two primers designed on exon 2 of theleptin gene, 5⬘-TTTGTCCAAGATGGACCAGACT-3⬘ and 5⬘-CAGGGAGCAGCTCTTGGA-3⬘, were digested with DdeI restriction endonuclease (New England Biolabs), which cleaves only Lepob allele-derived
products. The digested fragments were separated on a 2.5% NuSieve 3:1
agarose gel (BioWhittaker Molecular Applications, Rockland, ME). PCR
protocol was 35 cycles of 94 °C for 1 min, 60 °C for 1 min, and 72 °C for
1 min.
Mice were housed in a temperature-controlled environment with a
12-h light/dark cycle and free access to water and a standard chow diet
(Oriental MF, Oriental Yeast, Tokyo, Japan) containing 60% carbohydrate, 13% fat, and 27% protein on a caloric basis. All experiments were
performed with 12-week-old mice. For the Lepob/ob ⫻ Srebp-1⫺/⫺ experiment, mice were refed for 12 h following a 24-h fast prior to sacrifice inorder to minimize the variation in dietary conditions. For the fasting
and refeeding study, mice were fasted for 24 h or refed for 12 h after
24-h starvation. All animals were sacrificed in an early phase of thelight cycle under anesthesia with diethyl ether.
Blood Chemistries and Liver Lipid Analyses—Enzymatic assay kits
were used for the determination of cholesterol (Determiner TC; cholesterol oxidase method; Kyowa Medex, Tokyo, Japan), triglycerides (TGLH; lipoprotein lipase method; Wako Chemicals, Tokyo, Japan), andnonesterified fatty acids (nonesterified fatty acid C; acyl-CoA oxidase
method; Wako Chemicals). Plasma glucose was measured by ANTSENSE II (Bayer Medical, Tokyo, Japan) based on the immobilized
glucose oxidase membrane/hydrogen peroxide electrode method.
Plasma insulin was determined by the mouse insulin enzyme-linked
immunosorbent assay kit (Wako Chemicals). The content of cholesterol
and triglycerides in liver was measured as described previously (25).
RNA Isolation and Northern Blotting—Total RNA from liver andepididymal fat pad was isolated with Trizol reagent (Invitrogen), and10-␮g RNA samples equally pooled from six mice of each genotype wererun on a 1% agarose gel containing formaldehyde and transferred to anylon membrane. The cDNA probes used were cloned as previously
described (15, 26). The probes were labeled with [␣-32P]dCTP using the
Megaprime DNA labeling system kit (Amersham Biosciences). The
membranes were hybridized with the radiolabeled probe in Rapid-hyb
Buffer (Amersham Biosciences) at 65 °C with the exception of TNF␣, forwhich ULTRAhyb hybridization buffer (Ambion) was used at 42 °C. The
membranes were washed in 0.1⫻ SSC, 0.1% SDS at 65 °C. Blots were
exposed to Eastman Kodak Co. XAR-5 film and the BAS imaging plate
for the BAS2000 BIO IMAGING ANALYZER (Fuji Photo Film, Tokyo,
Japan). The quantification results obtained with the BAS2000 systemwere normalized to the signal generated from 36B4 (acidic ribosomal
phosphoprotein P0) mRNA.
RESULTS

Effects of SREBP-1 Absence on Obesity, Insulin Resistance,and Fatty Liver in Lepob/ob Mice—To assess the potential effectsof SREBP-1 deficiency on obesity and its related syndromes, weintercrossed Lepob/ob and SREBP-1-null mice and obtained six
male and six female mice deficient in both leptin and SREBP-1(Lepob/ob ⫻ Srebp-1⫺/⫺) in the C57BL/6J background. The frequency for obtaining the Lepob/ob ⫻ Srebp-1⫺/⫺ progeny was

FIG. 1. Effects of SREBP-1 disruption on fatty livers in Lepob/ob
mice. Liver weight/body weight ratio (a) and liver triglyceride content(b) in wild-type (䡺) C57BL/6J mice, Lepob/ob (f), Lepob/ob ⫻ Srebp-1⫹/⫺;;;;;;(o), and Lepob/ob ⫻ Srebp-1⫺/⫺ (;;;;;;) mice. Bars, S.E. for each group. pvalues (Student’s t test) indicate differences between Lepob/ob (n ⫽ 6)
ob/ob
⫺/⫺
and Lep⫻ Srebp-1
mice (n ⫽ 6).

FIG. 2. Effects of SREBP-1 deletion on the hepatic gene expression in Lepob/ob mice. Northern blot analysis of various genes including lipogenic and glycolytic enzymes in liver is shown. Lanes (left toright) show wild type (WT), Lepob/ob, Lepob/ob ⫻ Srebp-1⫹/⫺, and Lepob/ob⫻ Srebp-1⫺/⫺. Total RNA (10 ␮g) pooled equally from six mice wassubjected to Northern blotting, followed by hybridization with the indicated cDNA probes. FAS, fatty acid synthase; SCD, stearoyl-CoA
desaturase; GPAT, glycerol-3-phosphate acyltransferase; ACL, ATP citrate lyase; S14, Spot 14; HMGCoAsyn, hydroxymethylglutaryl-CoA
synthase; G6PD, glucose-6-phosphate dehydrogenase; PK, pyruvate kinase; GK, glucokinase. A cDNA probe for 36B4 (acidic ribosomal phosphoprotein P0) was used to confirm equal loading. *, the aberrant
messenger RNA from the disrupted Srebp-1 allele that encodes a truncated protein that is null for transcriptional activity. The results ofquantification by the BAS imaging plate are shown in Table II.

3-fold lower than expected, presumably due to the partial embryonic lethality of SREBP-1-null mice (24). This lower proportion of Srebp-1⫺/⫺ homozygotes was similar at all Lep genotypes, indicating that there were no lethal interactions between
the two gene deficiencies. These Lepob/ob ⫻ Srebp-1⫺/⫺ mice
showed no significant difference in body weight throughout theSREBP-1 Disruption in Lepob/Lepob Mice

19355

TABLE II
Quantification of gene expression levels in livers from wild-type, Lepob/ob, Lepob/ob ⫻ Srebp-1⫹/⫺, and Lepob/ob ⫻ Srebp-1⫺/⫺ mice by
Northern blot analysis shown in Fig. 2
Values are the relative ratio of each signal versus the corresponding Lepob/ob mice corrected for the signal from 36B4 (acidic ribosomal
phosphoprotein P0) as loading control. * and **, significance versus Lepob/ob at p ⬍ 0.05 and p ⬍ 0.01 (Student’s t test), respectively, determinedby another Northern blot analysis quantifying gene expression levels in individual mice. ND, not detectable. FAS, fatty acid synthase; SCD1,
stearoyl-CoA desaturase-1; GPAT, glycerol-3-phosphate acyltransferase; ACL, ATP citrate lyase; S14, Spot 14; HMGCoAsyn, hydroxymethylglutaryl-CoA synthase; G6PD, glucose-6-phosphate dehydrogenase; PK, pyruvate kinase; GK, glucokinase.
Genotype

Wild type

Lepob/ob

Lepob/ob ⫻ Srebp-1⫹/⫺

Lepob/ob ⫻ Srebp-1⫺/⫺

SREBP1
FAS
SCD1
GPAT
ACL
S14
SREBP2
HMGCoAsyn
G6PD
PK
GK

0.83
0.77
0.29*
0.63
1.06
0.70
0.94
0.73
1.40
0.74
0.65

1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00

0.53**
0.90
1.34
0.86
0.98
0.92
1.30
2.06*
0.83
1.16
1.05

ND
0.72**
0.82*
0.31**
0.70*
0.44**
1.98**
2.63**
1.11
0.80
1.08

FIG. 3. Effects of SREBP-1 absence on the mRNA expression ofvarious genes in adipose tissue from Lepob/ob mice. Northern blot
analysis of various genes including lipogenic enzymes in adipose tissue
is shown. Lanes (left to right) show wild type (WT), Lepob/ob, Lepob/ob ⫻Srebp-1⫹/⫺, and Lepob/ob ⫻ Srebp-1⫺/⫺. Total RNA (10 ␮g) pooled equallyfrom six mice was subjected to Northern blotting, followed by hybridization with the indicated cDNA probes. FAS, fatty acid synthase; SCD,
stearoyl-CoA desaturase; GPAT, glycerol-3-phosphate acyltransferase;
ACL, ATP citrate lyase; S14, Spot 14; HMGCoAsyn, hydroxymethylglutaryl-CoA synthase; LPL, lipoprotein lipase. A cDNA probe for 36B4
(acidic ribosomal phosphoprotein P0) was used to confirm equal loading. *, the aberrant messenger RNA from the disrupted Srebp-1 allele
that encodes a truncated protein. The results of quantification by the
BAS imaging plate are shown in Table III.

study period (until 12 weeks of age) or in epididymal orparametrial fat pad weight compared with Lepob/ob mice ofeither sex (Table I). In addition, the plasma glucose and insulin
concentration of Lepob/ob ⫻ Srebp-1⫺/⫺ mice were elevated tosimilar levels as those of Lepob/ob controls, suggesting that they
had an equal level of insulin resistance irrespective of their
Srebp-1 genotype. These results indicated that SREBP-1 absence had little influence on the development of obesity andinsulin resistance originating from leptin deficiency. Histological examination of the adipose tissue from Lepob/ob ⫻ Srebp1⫺/⫺ mice revealed no change in adipocyte hypertrophy ascompared with Lepob/ob mice (data not shown). Plasma cholesterol levels of double homozygotes were significantly lower
than those of Lepob/ob mice. Neither plasma triglyceride nor
nonesterified fatty acid was significantly altered by SREBP-1
disruption.
In contrast, the adiposity of Lepob/ob mouse liver was greatly
influenced by the Srebp-1 genotype. The triglyceride content in
the livers of Lepob/ob ⫻ Srebp-1⫺/⫺ mice was less than one-halfthat of Lepob/ob mice, and the Srebp-1 heterozygotes showed anintermediate value (Fig. 1b). The total liver weight of Lepob/ob ⫻Srebp-1⫺/⫺ mice was also decreased in comparison with
Lepob/ob controls (Fig. 1a), partly due to the diminished triglyceride accumulation. Cholesterol content in the livers of Lepob/ob⫻ Srebp-1⫺/⫺ mice was higher than in Lepob/ob mice, although
no statistical significance was observed.
We concluded from these findings that the absence of
SREBP-1 had no effect on the obesity and insulin resistance ofLepob/ob mice; however, it did ameliorate triglyceride accumulation in liver.
Mechanisms by Which SREBP-1 Disruption Attenuates Fatty
Liver in Lepob/ob Mice—To elucidate the underlying mechanisms for the amelioration of fatty livers by SREBP-1 knockoutin Lepob/ob mice, we evaluated the hepatic mRNA expression ofvarious lipogenic enzymes by Northern blot analysis (Fig. 2,
Table II). Total RNA was extracted from the livers of six miceof each genotype in a fed state. The mRNA abundance of
SREBP-1 was reduced in Lepob/ob ⫻ Srebp-1⫹/⫺ and was completely abolished in Lepob/ob ⫻ Srebp-1⫺/⫺ mice. The protein
product of the aberrant mRNA (denoted by asterisk in Figs.
2– 4) from the disrupted allele was previously reported to beinactive as a transcription factor (15, 24). By Northern blot
analysis, we were able to show that the mRNA levels of various
lipogenic enzymes such as fatty acid synthase, stearoyl-CoA
desaturase 1, glycerol-3-phosphate acyltransferase, ATP citrate lyase, and spot 14 were decreased by SREBP-1 deletion in
Lepob/ob mice livers. Meanwhile, mRNA levels for hydroxymethylglutaryl-CoA synthase, a key enzyme of cholesterol biosynthesis, and its controlling transcription factor, SREBP-2,
were reciprocally increased in Lepob/ob ⫻ Srebp-1⫺/⫺ mice.
These differences were confirmed to be statistically significant
by another Northern blot analysis quantifying gene expression
levels in individual animals. The expression levels of glucokinase and pyruvate kinase, major glycolytic enzymes, were notaltered by SREBP-1 disruption.
Distinct Influence of SREBP-1 Absence on Lipogenesis in
Adipose Tissue—While the adiposity of liver was significantly
decreased in the Lepob/ob⫻Srebp-1⫺/⫺ mice, adipose tissue mass
was unchanged. To investigate the mechanism by which thisoccurs, we evaluated the mRNA levels of various lipogenic
genes in the adipose tissue of mice from each group by Northern blot analysis (Fig. 3, Table III). Quite unexpectedly, themRNA abundance of lipogenic enzymes was markedly suppressed in adipose tissue of Lepob/ob mice compared with wildtype. Consistently, the mRNA level of SREBP-1 was also profoundly reduced in Lepob/ob mouse adipose tissue. The
disruption of SREBP-1 did not further decrease the lipogenic
gene expression in adipose tissue of Lepob/ob mice at all.

SREBP-1 Disruption in Lepob/Lepob Mice

19356

TABLE III
Quantification of gene expression levels in adipose tissue from wild-type, Lepob/ob, Lepob/ob ⫻ Srebp-1⫹/⫺, and Lepob/ob ⫻ Srebp-1⫺/⫺ mice by
Northern blot analysis shown in Fig. 3
Values are the relative ratio of each signal versus the corresponding Lepob/ob mice corrected for the signal from 36B4 (acidic ribosomal
phosphoprotein P0) as loading control. * and **, significance versus Lepob/ob at p ⬍ 0.05 and p ⬍ 0.01 (Student’s t test), respectively, determinedby another Northern blot analysis quantifying gene expression levels in individual mice. ND, not detectable. FAS, fatty acid synthase; SCD,
stearoyl-CoA desaturase-1; GPAT, glycerol-3-phosphate acyltransferase; ACL, ATP citrate lyase; S14, Spot 14; HMGCoAsyn, hydroxymethylglutaryl-CoA synthase; G6PD, glucose-6-phosphate dehydrogenase; PK, pyruvate kinase; GK, glucokinase.
Genotype

Wild type

Lepob/ob

Lepob/ob ⫻ Srebp-1⫹/⫺

Lepob/ob ⫻ Srebp-1⫺/⫺

SREBP1
FAS
SCD1
GPAT
ACL
S14
HMGCoAsyn
PPAR␥
TNF␣
LeptinLPL

4.04**
10.83**
0.86
3.49**
10.32**
6.49**
0.75
1.53*
0.39*
0.11**
0.70

1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00

0.71**
1.18
0.95
1.21
1.03
0.76
1.51*
1.07
1.10
1.07
1.03

ND
1.55
0.96
1.10
1.63
1.57
2.49**
1.14
1.08
0.91
0.98

FIG. 4. Effects of SREBP-1 absence on mRNA levels of leptin
and lipoprotein lipase genes in the adipose tissue of lean mice.
Northern blot analysis of leptin and lipoprotein lipase (LPL) genes inadipose tissue from wild-type (WT) and Srebp-1⫺/⫺ (KO) mice (four malemice each) was conducted in a fasted (left two lanes) or refed state in theLep⫹/Lep⫹ background. Total RNA (10 ␮g) pooled equally from four
mice was run in each lane. A cDNA probe for 36B4 (acidic ribosomal
phosphoprotein P0) was used to confirm equal loading. *, the aberrant
messenger RNA from the disrupted Srebp-1 allele that encodes a truncated protein.

Effects of SREBP-1 Disruption on Markers for Insulin Resistance—Several adipocyte-derived genes such as tumor necrosis
factor ␣ (TNF␣) and peroxisome proliferator-activated receptor
␥ (PPAR␥) have been implicated to be related to insulin resistance in adipose tissue (27), and their mRNA levels were also
examined in our study. We demonstrated that the expression of
PPAR␥, a nuclear receptor for thiazolidinedions, insulin-sensitizing drugs, was decreased, whereas TNF␣, which has beensuggested to be a causal cytokine for insulin resistance, wasincreased in the adipose tissue of Lepob/ob mice compared withwild type (Fig. 3, Table III), both of which reflect the insulinresistant state of Lepob/ob mice. However, the comparison of
Lepob/ob and Lepob/ob ⫻ Srebp-1⫺/⫺ mice revealed that neither
PPAR␥ nor TNF␣ was altered by the absence of SREBP-1 inthe Lepob/ob mouse adipose tissue. These data provided further
evidence that SREBP-1 is not involved in the insulin resistanceof Lepob/ob mice. The expression levels of lipoprotein lipase (17,28) and leptin (29) that had been reported to be regulated by
SREBP-1 were also independent of Srebp-1 genotype, which
held true again in the Lep⫹/Lep⫹ background (Fig. 4).

FIG. 5. Comparison of refeeding responses in lipogenic genes
between liver and adipose tissue from Lepob/ob mice. Northernblot analysis of lipogenic enzymes in liver (a) and adipose tissue (b) isshown. Wild-type (WT) and Lepob/ob (OB) mice (six male mice each) arecompared in a fasted (left two lanes) or refed state. Total RNA (10 ␮g)pooled equally from six mice was run in each lane. FAS, fatty acidsynthase; ACL, ATP citrate lyase; S14, Spot 14; HMGCoAsyn, hydroxymethylglutaryl-CoA synthase. A cDNA probe for 36B4 (acidic
ribosomal phosphoprotein P0) was used to confirm equal loading.

Defective Responses of Lepob/ob Lipogenic Enzymes in Adipose Tissue to Dietary Manipulation—Lipogenic enzymes are
known to be markedly induced in liver and adipose tissue when
animals are refed after starvation. To further study the unexpected suppression of lipogenic genes in Lepob/ob mouse adipose
tissue, we evaluated the refeeding response in the adipose
tissue of wild-type and Lepob/ob mice (Fig. 5). Livers from refedwild-type and Lepob/ob mice displayed a similar extent of induction of all lipogenic genes including the Srebp-1 gene. In contrast, the mRNA levels of SREBP-1 and lipogenic enzymes in
the adipose tissue stayed markedly repressed in the Lepob/ob
mice even after refeeding, whereas wild-type adipose tissue
showed robust refeeding responses. These data demonstrated
that the adipose tissue of Lepob/ob mice had dysregulation of
lipogenic gene expression in a fed state, whereas the liver ofLepob/ob mice was normal in the refeeding responses.
DISCUSSION

The current study clearly demonstrates that SREBP-1 plays
a crucial role in the development of fatty livers in Lepob/ob mice.
The disruption of SREBP-1 caused a significant reduction in
hepatic expression of a battery of lipogenic genes and prevented fatty livers in Lepob/ob mice, indicating that SREBP-1
controls triglyceride accumulation in the liver by regulating theSREBP-1 Disruption in Lepob/Lepob Mice
expression levels of lipogenic enzymes. These data are in accordance with previous reports that the overexpression of
SREBP-1 induced lipogenic enzymes and resulted in fatty livers in several mouse models including SREBP-1a and -1c transgenic mice (12, 23, 26). It can be concluded, therefore, that
SREBP-1 is a key transcription factor that nutritionally regulates hepatic gene expression of lipogenic enzymes and triglyceride deposition in the liver.
The observations from adipose tissue showed a remarkable
contrast to those from liver. We demonstrated from the comparison of wild-type and Lepob/ob mice that the mRNA levels of
lipogenic enzymes and adipose tissue mass were not correlated,suggesting that lipogenesis in adipose tissue is not the primarycause of obesity in Lepob/ob mice. The lower expression of
SREBP-1 and lipogenic enzymes in the Lepob/ob mouse adipose
tissue as compared with those in wild-type has also been described elsewhere as a part of the microarray analysis (30, 31).
The difference was more pronounced in our experiments thatwere performed in a refed state (Fig. 5). It has been shown by
enzymatic assay experiments that lipogenesis in Lepob/obmouse adipose tissue is elevated in the early dynamic phase
until 7– 8 weeks of age and thereafter suppressed in the latestatic phase when insulin resistance becomes evident (32, 33).
In contrast, hepatic lipogenesis in Lepob/ob mice remains consistently higher than in wild-type animals. Therefore, lipogenesis is not likely to be important in the sustained hypertrophyof adipocytes in older Lepob/ob mice. These results can be explained by the fact that adipose mass is related not only to denovo fatty acid synthesis but also to fatty acid intake mediatedthrough lipoprotein lipase (34). In our data, the expression of
lipoprotein lipase was not affected by SREBP-1 absence, which
could be another reason for sustained obesity in the doubly
mutant mice. Adipocyte hypertrophy can be also influenced bylipolysis through the action of hormone-sensitive lipase (35).
We conclude from these results that the inherent suppression of SREBP-1 in Lepob/ob mouse adipose tissue was theprimary cause of the ineffectiveness of SREBP-1 disruption on
obesity. Another factor that might explain the phenotypic discrepancy between liver and adipose tissue in the Lepob/ob ⫻Srebp-1⫺/⫺ mice is the different contribution of SREBP-1 to theregulation of lipogenesis in the two organs (15); the disruptionof SREBP-1 strongly suppressed the refeeding responses oflipogenic enzymes in the liver, whereas their expression wasless influenced by SREBP-1 levels in the adipose tissue. It is
possible that there are other specific transcription factors that
regulate lipogenesis in adipocytes and that SREBP-1 contribution is of minor importance in this tissue (15).
We demonstrated that the adipose tissue of older Lepob/ob
mice could not respond fully to the refeeding manipulation,
whereas the livers responded normally at least with respect tolipogenesis. Given that the refeeding response of lipogenesis
highly depends upon insulin action, it could be said that hyperinsulinemia in Lepob/ob mice could overcome insulin resistance to lipogenesis in the liver but could not in the adipose
tissue. This is consistent with previous reports that the adipose
tissue was more affected in an insulin-resistant state of Lepob/ob
mice than the liver where lipid synthesis remained sensitive toinsulin stimulation throughout life (6).
It has been reported that nonalcoholic fatty liver disease isassociated with insulin resistance and hyperinsulinemia evenin lean subjects with normal glucose tolerance (36). Fatty liverdisease and insulin resistance in these patients might represent an initial stage of the metabolic syndrome X (37, 38). In
this study, we were able to segregate fatty liver disease frominsulin resistance syndromes by the disruption of SREBP-1, an
indication that excess triglyceride accumulation in the liver is19357

not a cause but rather the result of insulin resistance andhyperinsulinemia.
In summary, we demonstrated that the absence of SREBP-1
attenuated fatty livers but not obesity or insulin resistance in
Lepob/ob mice. It was revealed that SREBP-1 plays a crucial rolein the regulation of lipogenic gene expression and triglyceride
accumulation in the liver. The ineffectiveness of SREBP-1 disruption on obesity presumably resulted from the decreased
expression of SREBP-1 in Lepob/ob mouse adipose tissue. The
data also suggested that lipogenesis is not a determinant ofobesity in Lepob/ob mice.
Acknowledgments—We are grateful to Drs. Michael S. Brown andJoseph L. Goldstein for continuous support for the project. We appreciate Dr. K. Komeda for support in maintaining mouse colonies.
REFERENCES
1. Kuczmarski, R. J., Flegal, K. M., Campbell, S. M., and Johnson, C. L. (1994)
J. Am. Med. Assoc. 272, 205–211
2. Saltiel, A. R. (2001) Cell 104, 517–529
3. Ingalls, A. M., Dickie, M. M., and Snell, G. D. (1950) J. Hered. 41, 317–318
4. Mayer, J., Bates, M. W., and Dickie, M. M. (1951) Science 113, 746 –747
5. Herberg, L., and Coleman, D. L. (1977) Metabolism 26, 59 –99
6. Bray, G. A., and York, D. A. (1979) Physiol. Rev. 59, 719 – 809
7. Zhang, Y., Proenca, R., Maffei, M., Barone, M., Leopold, L., and Friedman,
J. M. (1994) Nature 372, 425– 432
8. Montague, C. T., Farooqi, I. S., Whitehead, J. P., Soos, M. A., Rau, H.,
Wareham, N. J., Sewter, C. P., Digby, J. E., Mohammed, S. N., Hurst, J. A.,
Cheetham, C. H., Earley, A. R., Barnett, A. H., Prins, J. B., and O’Rahilly,
S. (1997) Nature 387, 903–908
9. Clandinin, M. T., Cheema, S., Pehowich, D., and Field, C. J. (1996) Lipids 31,
Suppl. 2, 13–22
10. Yokoyama, C., Wang, X., Briggs, M. R., Admon, A., Wu, J., Hua, X., Goldstein,
J. L., and Brown, M. S. (1993) Cell 75, 187–197
11. Brown, M. S., and Goldstein, J. L. (1997) Cell 89, 331–340
12. Shimano, H., Horton, J. D., Shimomura, I., Hammer, R. E., Brown, M. S., andGoldstein, J. L. (1997) J. Clin. Invest. 99, 846 – 854
13. Horton, J. D., Shimomura, I., Brown, M. S., Hammer, R. E., Goldstein, J. L.,
and Shimano, H. (1998) J. Clin. Invest. 101, 2331–2339
14. Shimomura, I., Shimano, H., Korn, B. S., Bashmakov, Y., and Horton, J. D.
(1998) J. Biol. Chem. 273, 35299 –35306
15. Shimano, H., Yahagi, N., Amemiya-Kudo, M., Hasty, A. H., Osuga, J., Tamura,Y., Shionoiri, F., Iizuka, Y., Ohashi, K., Harada, K., Gotoda, T., Ishibashi,S., and Yamada, N. (1999) J. Biol. Chem. 274, 35832–35839
16. Tontonoz, P., Kim, J. B., Graves, R. A., and Spiegelman, B. M. (1993) Mol. Cell.
Biol. 13, 4753– 4759
17. Kim, J. B., and Spiegelman, B. M. (1996) Genes Dev. 10, 1096 –1107
18. Horton, J. D., Bashmakov, Y., Shimomura, I., and Shimano, H. (1998) Proc.
Natl. Acad. Sci. U. S. A. 95, 5987–5992
19. Osborne, T. F. (2000) J. Biol. Chem. 275, 32379 –32382
20. Foretz, M., Guichard, C., Ferre, P., and Foufelle, F. (1999) Proc. Natl. Acad.
Sci. U. S. A. 96, 12737–12742
21. Shimomura, I., Bashmakov, Y., Ikemoto, S., Horton, J. D., Brown, M. S., andGoldstein, J. L. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 13656 –13661
22. Flier, J. S., and Hollenberg, A. N. (1999) Proc. Natl. Acad. Sci. U. S. A. 96,
14191–14192
23. Shimomura, I., Bashmakov, Y., and Horton, J. D. (1999) J. Biol. Chem. 274,
30028 –30032
24. Shimano, H., Shimomura, I., Hammer, R. E., Herz, J., Goldstein, J. L., Brown,
M. S., and Horton, J. D. (1997) J. Clin. Invest. 100, 2115–2124
25. Yokode, M., Hammer, R. E., Ishibashi, S., Brown, M. S., and Goldstein, J. L.
(1990) Science 250, 1273–1275
26. Shimano, H., Horton, J. D., Hammer, R. E., Shimomura, I., Brown, M. S., andGoldstein, J. L. (1996) J. Clin. Invest. 98, 1575–1584
27. Spiegelman, B. M., and Flier, J. S. (1996) Cell 87, 377–389
28. Schoonjans, K., Gelman, L., Haby, C., Briggs, M., and Auwerx, J. (2000) J. Mol.
Biol. 304, 323–334
29. Kim, J. B., Sarraf, P., Wright, M., Yao, K. M., Mueller, E., Solanes, G., Lowell,
B. B., and Spiegelman, B. M. (1998) J. Clin. Invest. 101, 1–9
30. Soukas, A., Cohen, P., Socci, N. D., and Friedman, J. M. (2000) Genes Dev. 14,
963–980
31. Nadler, S. T., Stoehr, J. P., Schueler, K. L., Tanimoto, G., Yandell, B. S., andAttie, A. D. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 11371–11376
32. Hems, D. A., Rath, E. A., and Verrinder, T. R. (1975) Biochem. J. 150, 167–173
33. Kaplan, M. L., and Leveille, G. A. (1981) Am. J. Physiol. 240, E101–E107
34. Weinstock, P. H., Levak-Frank, S., Hudgins, L. C., Radner, H., Friedman,
J. M., Zechner, R., and Breslow, J. L. (1997) Proc. Natl. Acad. Sci. U. S. A.
94, 10261–10266
35. 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
36. Lee, J. H., Rhee, P. L., Lee, J. K., Lee, K. T., Kim, J. J., Koh, K. C., Paik, S. W.,
Rhee, J. C., and Choi, K. W. (1998) Korean J. Intern. Med. 13, 12–14
37. Reaven, G. M. (1988) Diabetes 37, 1595–1607
38. Marchesini, G., Brizi, M., Morselli-Labate, A. M., Bianchi, G., Bugianesi, E.,
McCullough, A. J., Forlani, G., and Melchionda, N. (1999) Am. J. Med. 107,
450 – 455