THE JOURNAL OF BIOLOGICAL CHEMISTRY© 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 279, No. 20, Issue of May 14, pp. 20571–20575, 2004
Printed in U.S.A.

p53 Involvement in the Pathogenesis of Fatty Liver Disease*
Received for publication, January 27, 2004, and in revised form, February 24, 2004
Published, JBC Papers in Press, February 25, 2004, DOI 10.1074/jbc.M400884200

Naoya Yahagi‡, Hitoshi Shimano§¶, Takashi Matsuzaka§, Motohiro Sekiya‡, Yuho Najima‡,
Sachiko Okazaki‡, Hiroaki Okazaki‡, Yoshiaki Tamura‡, Yoko Iizuka‡, Noriyuki Inoue§,
Yoshimi Nakagawa§, Yoshinori Takeuchi§, Ken Ohashi‡, Kenji Harada‡, Takanari Gotoda‡,
Ryozo Nagai‡, Takashi Kadowaki‡, Shun Ishibashi‡, Jun-ichi Osuga‡, and Nobuhiro Yamada§From the ‡Department of Internal Medicine, Graduate School of Medicine, University of Tokyo, Tokyo 113-8655 and the§Department of Internal Medicine, Institute of Clinical Medicine, University of Tsukuba, Ibaraki 305-8575, Japan

Obesity is a major health problem in industrializedsocieties, and fatty liver disease (hepatic steatosis) is
common in obese individuals. Oxidative stress originating from increased intracellular levels of fatty acids has
been implicated as a cause of hepatocellular injury insteatosis, although the precise mechanisms remain to beelucidated. p53, widely known as a tumor suppressor,
has been shown often to be activated in stressed cells,
inducing cell cycle arrest or death. Here we demonstratethat p53 is involved in the molecular mechanisms ofhepatocellular injury associated with steatosis. We
found that p53 in the nucleus is induced in the liver fromtwo mouse models of fatty liver disease, ob/ob and atransgenic mouse model that overexpresses an active
form of sterol regulatory element-binding protein-1 inthe liver (TgSREBP-1), the one with obesity and theother without obesity. This activation of the p53 pathway leads to the elevation of p21 mRNA expression,
which can be considered an indicator of p53 activity,
because ob/ob mice lacking p53 generated by targeting
gene disruption exhibited the complete restoration of
the p21 elevation to wild type levels. Consistent withthese results, the amelioration of hepatic steatosis
caused by Srebp-1 gene disruption in ob/ob mice lowered
the p21 expression in a triglyceride content-dependent
manner. Moreover, p53 deficiency in ob/ob mice resulted
in a marked improvement of plasma alanine aminotransferase levels, demonstrating that p53 is involvedin the mechanisms of hepatocellular injury. In conclusion, we revealed that p53 plays an important role in thepathogenesis of fatty liver disease.

Obesity is frequently associated with a number of diverse
diseases, including type II diabetes, hyperlipidemia, and cardiovascular disease, and is a major health problem in industrialized societies (1, 2). Epidemiologic evidence suggests thatliver disease might be a major contributor to overall obesityrelated morbidity and mortality, affecting 70% of obese individuals (reviewed in Refs. 3 and 4).
The genetically obese ob/ob mice develop obesity, insulin
resistance, and glucose intolerance because of an inherited* This work was supported in part by a grant from the Research
Fellowships of the Japan Society for the Promotion of Science (to N. Y.).
The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate 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. Fax: 81-298-63-2081;
E-mail: shimano-tky@umin.ac.jp.
This paper is available on line at http://www.jbc.org

deficiency of the appetite-suppressing hormone, leptin (5–9).
The absence of leptin presents the most severe obesity known
in both rodents and humans (10) and provides a good model ofobesity and its related syndromes, including fatty liver disease.
It has been documented that lipogenesis in the liver is increased in ob/ob mice (7, 8). We have demonstrated previouslythat the disruption of sterol regulatory element-binding protein-1 (SREBP-1),1 the key transcriptional regulator of lipogenesis (reviewed in Refs. 11 and 12), leads to marked amelioration of hepatic steatosis (13, 14).
The pathogenesis of liver disease associated with obesity hasremained poorly understood. Although fatty liver is typicallybenign, it is believed that in a subset of individuals gradualprogression from hepatic steatosis to steatohepatitis and eventually to cirrhosis can occur. Since liver cell damage is reversible by caloric restriction at least at the early stages of thedisease, it is postulated that nutritional overload plays a pivotal role in the mechanisms of hepatocellular injury. Notably,
fatty liver disease also develops without obesity in some mousemodels, when SREBP-1 expression and thereby lipogenesis areelevated in the liver (15, 16). Thus, the retention of triglycerides within hepatocytes is a prerequisite for the development of
fatty liver disease, exerting toxic effects on hepatocytes. As amechanism by which overnutrition causes liver cell damage,oxidative stress originating from increased intracellular levelsof fatty acids is considered to be involved (3, 4), although theseconcepts remain largely hypothetical.
The p53 gene was the first tumor-suppressor gene to be identified and has been found to be inactivated in most human cancers (17). The p53 protein prevents division of stressed cells or
causes them to undergo programmed cell death (apoptosis). For
example, ␥-irradiation activates p53 to turn on the transcriptionof p21Waf1/CIP1, which binds to and inhibits cyclin-dependent
kinases, thus blocking the G1-S and G2-to-mitosis transitions.
The stresses that activate p53 are diverse, ranging from DNA
damage to oxidative stress, hypoxia, and heat shock (18). Thus,
p53 has been thought to be a guardian against cellular stresses
and has been extensively studied and well established as a tumor
suppressor. However, much less is known about other roles ofp53 beyond tumor suppression. Notably recent reports (19 –21)
document that p53 is involved in the molecular mechanisms ofsome types of hepatocellular injury.
We have reported previously (22) that p53 is activated inadipocytes of ob/ob mice, being involved in the mechanisms ofdysregulated gene expression in adipose tissue of obese animals. These mice have largely excessive fat deposition in adi1
The abbreviations used are: SREBP-1, sterol regulatory elementbinding protein-1; ALT, alanine aminotransferase; TG, triglyceride;
WT, wild type.

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p53 Involvement in Fatty Liver Disease

FIG. 1. p53 activation in the nucleus of ob/ob mouse liver. a,
Northern blot; b, Western blot analysis of p53 in WT and ob/ob (OB)mouse liver. a, total RNA (10 ␮g) pooled equally from three male micefor each group was subjected to Northern blotting, followed by hybridization with the indicated cDNA probes. A cDNA probe for 36B4 (acidic
ribosomal phosphoprotein P0) was used to confirm equal loading. b,
aliquots of nuclear protein (20 ␮g) extracted from livers equally pooled
from three male mice for each group were subjected to SDS-PAGE.
Immunoblotting (IB) using antibody against full-length p53 protein (leftpanel) and against phosphorylated Ser-15 (right panel) is shown. Seriesof experiments were performed twice for the same number of animals,
and the data from a representative experiment are shown.

FIG. 2. p53 activation in the nucleus of TgSREBP-1 mouse
liver. a, Northern blot; b, Western blot analysis of p53 in WT and
TgSREBP-1 (Tg) mouse liver (three male mice each). a, total RNA (10␮g) was subjected to Northern blotting, followed by hybridization with
the indicated cDNA probes. A cDNA probe for 36B4 (acidic ribosomal
phosphoprotein P0) was used to confirm equal loading. b, aliquots ofliver nuclear protein (20 ␮g) were subjected to SDS-PAGE. Immunoblotting (IB) using antibody against full-length p53 protein (left panel)and against phosphorylated Ser-15 (right panel) is shown. Series ofexperiments were performed twice for the same number of animals, andthe data from a representative experiment are shown.

pocytes and hepatocytes. If overloaded fat stresses adipocytes
thereby activating the p53 pathway, the same mechanism
could be provoked in the steatotic hepatocytes of obese animals,presumably leading to hepatocellular injury.
Based on these considerations, we hypothesized that steatotic hepatocytes are under various stresses that induce p53,
which gives rise to liver cell damage and plays an importantrole in the development of fatty liver disease. In our present
study, we demonstrate that nuclear p53 is induced in the liverfrom two mouse models of fatty liver disease, one with obesity
and the other without obesity, elevating p21 mRNA expression
as an indicator of its activity and that, as we expect, p53 isinvolved in the mechanisms of hepatocellular injury accompanied by steatosis.
EXPERIMENTAL PROCEDURES

Animals—Lep⫹/ob-C57BL/6J and p53⫹/⫺-C57BL/6J (23) mice werepurchased from The Jackson Laboratories (Bar Harbor, ME). Mice
deficient in SREBP-1 were established as described previously (24) andwere backcrossed 6 times onto the C57BL/6J background. These mice
were intercrossed to produce ob/ob mice lacking p53 (ob/ob ⫻ p53⫺/⫺)or SREBP-1 (ob/ob ⫻ Srebp-1⫺/⫺) as described previously (13, 22).
Genotypes at the p53 loci were determined by PCR (22); those at theleptin loci were by a PCR-based restricted fragment length polymorphism analysis (13), and those at the SREBP-1 loci were by Southernblot analysis with BamHI digestion (24). SREBP-1a transgenic miceoverexpressing amino acids 1– 460 of human SREBP-1a under controlof the rat phosphoenolpyruvate carboxykinase promoter (TgSREBP-1)
were made as described previously (15). Genotypes at the transgenic
loci were determined by PCR, and heterozygous mice were used. 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). All experiments were
performed with 12-week-old male mice. All animals were sacrificed inan early phase of the light cycle.
Nuclear Protein Extraction and Western Blotting—Nuclear extracts
from mice livers were prepared as described previously (25). Aliquots ofnuclear protein (20 ␮g) were subjected to SDS-PAGE. p53 was detected

FIG. 3. p53 activation in TgSREBP-1 mouse liver causes elevation of p53-regulated genes. Northern blot analysis of p53 downstream genes in livers from WT and TgSREBP-1 (Tg) mice (three malemice each) is shown. Total RNA (10 ␮g) was subjected to Northernblotting, followed by hybridization with the indicated cDNA probes. A
cDNA probe for 36B4 (acidic ribosomal phosphoprotein P0) was used toconfirm equal loading. Series of experiments were performed twice forthe same number of animals, and the data from a representative experiment are shown.
using a 1:500 dilution of anti-p53FL (sc-6243, Santa Cruz Biotechnology). The phosphorylation at Ser-15 of p53 was detected using a 1:1000
dilution of anti-p53 Ser-15 (9284S, New England Biolabs). Bound antibodies were detected with a horseradish peroxidase-coupled anti-rabbit
IgG secondary antibody (Amersham Biosciences) and visualized using
SuperSignal West Dura chemiluminescent substrates (Pierce).
RNA Isolation and Northern Blotting—Total RNA from mouse liver
was extracted using Trizol reagent (Invitrogen), and 10 ␮g of RNA
samples equally pooled from each group (n ⫽ 3– 6) were run on a 1%

p53 Involvement in Fatty Liver Disease

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FIG. 4. p21 mRNA expression as anindicator of p53 activity parallels
liver triglyceride contents. a, Northern blot analysis of p53-regulated genes
in livers from WT and ob/ob mice (sixmale mice each) lacking SREBP-1 or p53,
whose genotypes are indicated in thepanel. Total RNA (10 ␮g) was subjected to
Northern blotting, followed by hybridization with the indicated cDNA probes. A
cDNA probe for 36B4 (acidic ribosomal
phosphoprotein P0) was used to confirm
equal loading. The quantification results
obtained with BAS2000 system were normalized to the signal generated from
36B4 (acidic ribosomal phosphoprotein
P0) mRNA, and the fold changes against
control are displayed below each blot. b,
relationship between liver triglyceride(TG) contents (on horizontal axis) and p21
mRNA expression level (on vertical axis;
the same data as shown above) is plotted.
Genotypes are indicated in the panel. The
regression line among the points for wild
type, ob/ob, ob/ob⫻Srebp-1⫹/⫺, andob/ob ⫻ Srebp-1⫺/⫺ is inserted. Bars, S.E.

agarose gel containing formaldehyde and transferred to a nylon membrane. The cDNA probes were cloned as described previously (15, 22,26). The probes were labeled with [␣-32P]dCTP using 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 p53, for which 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 Kodak
XAR-5 film.
Blood Chemistries and Liver Lipid Analyses—Plasma alanine aminotransferase (ALT) activities were measured on an automated analyzer
at a clinical chemistry laboratory (Special Reference Laboratories, Tokyo, Japan). The contents of triglycerides in liver were measured as
described previously (27). Enzymatic assay kit was used for the determination of triglycerides (LH, Wako Pure Chemicals, Tokyo, Japan).
RESULTS

p53 Activation in Two Models of Fatty Livers, ob/ob and
TgSREBP-1 Mice—In our first series of experiments, we examined the p53 expression both at mRNA and protein levels in the
nucleus from livers of ob/ob mice. These mice present amarked increase in hepatic triglyceride contents, i.e. fatty livers (hepatic steatosis), and are a good model for fatty liverdisease associated with obesity. We extracted nuclear proteinsfrom control and ob/ob mice livers, and we analyzed them byWestern blotting with anti-p53 and anti-phosphorylated p53 at
Ser-15 antibodies. As shown in Fig. 1, we demonstrated thatob/ob mouse liver had higher levels of p53 protein in the
nucleus than wild type. Consistently, an active form of p53,
phosphorylated at Ser-15, was also up-regulated in ob/ob livers(Fig. 1). In contrast, p53 mRNA level was not elevated in ob/oblivers.
We also performed these analyses of p53 expression in
SREBP-1 transgenic (TgSREBP-1) mice. This is another modelof fatty liver but does not display systemic obesity. Similarly to
ob/ob mice, TgSREBP-1 mice showed elevated expression ofp53 protein with no increase in mRNA expression levels (Fig.
2). Phosphorylation of p53 at Ser-15 was also increased inparallel.
p53 Activation in Fatty Liver Causes Elevation of p53-regulated Genes—To explore the effects of p53 activation in the
nucleus from fatty livers, we further examined the expression
profile of p53 downstream genes such as p21, Mdm-2, and Bax

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p53 Involvement in Fatty Liver Disease␣ in these animals. As shown in Fig. 3, these p53-regulated
genes were elevated in the livers of TgSREBP-1 mice. In contrast, only p21 was elevated in ob/ob livers, whereas Mdm-2
and Bax ␣ were not altered in mRNA expression levels (Fig.
4a). The mRNA expression of Fas, another p53 downstream
gene, was not increased in either mouse model. These datasuggest that p53 activation causes the up-regulation of p21 as
its target gene in livers of ob/ob and TgSREBP-1 mice, andthat the mRNA expression level of p21 is a good indicator of p53
activity in fatty livers.
p21 mRNA Expression as an Excellent Indicator of p53 Activity—To validate that the elevation of p21 in fatty liver isreally caused by p53, we assessed the effects of p53 deficiency
in ob/ob mice. For this purpose, we intercrossed ob/ob andp53-null mice, and we obtained six male mice deficient in both
leptin and p53 (ob/ob ⫻ p53⫺/⫺) in the C57BL/6J background.
These doubly homozygous mice were equally obese comparedwith ob/ob mice as we reported previously (22). The Northernblot analysis on livers from these mice revealed that the disruption of p53 completely abolished the up-regulation of p21
mRNA expression observed in ob/ob mice to the same levels as
in wild type (Fig. 4, a and b). Meanwhile, the mRNA expression
levels of Mdm-2, Bax ␣, and Fas were not influenced by p53
disruption. These results established that p53 activation
caused the elevated expression of p21 in ob/ob livers and that
p21 mRNA level is a good indicator of p53 activity.
Disruption of SREBP-1 Ameliorates Fatty Livers andThereby Lowers p21 Expression—To clarify further the relationship between fatty livers and the p53-p21 pathway, we
analyzed ob/ob mice deficient in SREBP-1. These mice wereobtained by intercrossing of ob/ob and SREBP-1-null mice in
the C57BL/6J background (13), and as we reported previously,
the double homozygotes exhibit a marked improvement of fatty
livers. As expected, p21 mRNA expression in livers of double
homozygotes (ob/ob ⫻ Srebp-1⫺/⫺; n ⫽ 6) was decreased tonear wild type levels, and heterozygotes (ob/ob ⫻ Srebp-1⫹/⫺)
were at an intermediate level (Fig. 4, a and b), so the improvement of hepatic steatosis by Srebp-1 gene disruption in ob/ob
mice completely paralleled the decrease in p21 expression.
These results demonstrate that amelioration of fatty livers
lowers the elevated expression of p21.
p53 Disruption Alleviates Liver Injury of ob/ob Mice—ALT isa liver-specific enzyme that is released into the blood when
hepatocytes are damaged. Therefore, plasma ALT levels arewidely used as an excellent marker to evaluate liver injury, and
it is well known that they are elevated in humans and animals
with fatty livers including ob/ob mice. To estimate the degreeof liver cell damage in ob/ob mice lacking SREBP-1, we measured plasma ALT levels in these mice. As expected, ob/ob mice
without either allele of Srebp-1 gene (ob/ob ⫻ Srebp-1⫺/⫺)
showed decreased levels of ALT, and heterozygotes (ob/ob ⫻Srebp-1⫹/⫺) were at an intermediate position (Fig. 5a). These
values are completely in parallel with the hepatic triglyceride
contents, indicating that plasma ALT levels are a good marker
for hepatic steatosis accompanied by liver injury. Next weevaluated plasma ALT in ob/ob mice deficient in p53. Notably,
the double homozygotes (ob/ob ⫻ p53⫺/⫺), whose hepatic triglyceride contents were equal to those of ob/ob mice as shown in
Fig. 4b, exhibited marked reduction in plasma ALT levels compared with ob/ob mice. These findings demonstrate that p53mediated cytotoxicity plays an important role in the pathogenesis of liver injury concomitant with hepatic steatosis.
DISCUSSION

Our present studies clearly demonstrate that p53 is activated to induce p21 expression in two different models of hepatic steatosis, and that the p53 pathway is involved in the

FIG. 5. p53 disruption alleviates liver injury of ob/ob mice. a,plasma ALT activity in WT and ob/ob mice with various Srebp-1
genotype (⫹/⫹, ⫹/⫺, or ⫺/⫺ as indicated). Each group consists of six
male mice. p value was calculated by Student’s t test against ob/ob with
BP1⫹/⫹ mice. b, plasma ALT levels in ob/ob mice lacking p53 allele(six male mice each) are shown. Genotypes are as indicated. Mice were12-week old. Bars, S.E.

pathogenesis of fatty liver disease. Although others have reported that p53 activation itself coincides with fatty liver inhumans (28), this is the first report to describe the role of p53
in the mechanisms of hepatocellular injury accompanied byhepatic steatosis.
The mechanisms of hepatocellular injury associated withsteatosis have largely been unknown. It is postulated that
excess fat accumulation in the liver predisposes cells to hepatocellular injury, and that this is presumably caused by thecellular toxicity of excess free fatty acids, oxidative stress, andlipid peroxidation (3, 4). All of these can potentially activate thep53 pathway as causes of p53 activation are diverse, ranging
from DNA damage to oxidative stress and hypoxia (18). In
particular, oxidative stress is currently stated to be a central
mechanism of cytotoxicity in steatotic liver, and multiple potential sources of oxidative stress have been identified including cytochrome P4502E1 (CYP2E1), peroxisomal ␤-oxidation,
mitochondrial electron leak, and ceramide production (4, 29).
These oxidative stresses might possibly be involved in themechanisms of p53 activation observed in our current study.
Moreover, reactive oxygen species-induced cytokine release(transforming growth factor-␤, tumor necrosis factor-␣, or interleukin-8) that may contribute to the pathogenesis of liver
injury (19, 30) has also been shown to induce p53 activation(21, 31, 32).

p53 Involvement in Fatty Liver Disease
We have reported recently (22) that p53 is activated in adipocytes of ob/ob mice. It is very intriguing if one can assume
that some kind of stresses brought about by excess fat accumulation compel this activation and that the same mechanisms
could be turned on in case of steatotic hepatocytes. This analogy is also remarkable from the standpoint of the lipotoxicity
theory proposed by Unger (29), where it is assumed that in thepathogenesis of obesity triglycerides overaccumulate in various
tissues and exert toxic effects.
The molecular mechanism by which p53 causes hepatocellular injury also remains obscure. Bax protein is reported to be
implicated in the cytotoxic effects of p53 through transcriptional up-regulation (33) and is demonstrated to be increased in
ob/ob mouse liver (34). However, we detected no elevation ofBax mRNA expression in ob/ob liver nor reduction in ob/ob
mice lacking p53 (ob/ob ⫻ p53⫺/⫺), although it was induced in
TgSREBP-1 mouse liver. Fas mRNA expression was not increased, which is also reported to be a p53-regulated cell surface protein known to trigger apoptosis (35). These results are
not surprising because it is known that the p53 target genes
are widely divergent and tissue-dependent (36). As far as we
examined, the only gene found to be up-regulated by p53 was
p21, but it is not generally considered as a cytotoxic molecule.
The actual mediator of the cytotoxic action of p53 needs to be
identified in further investigations.
Our findings may have good clinical implication for a therapy
against fatty liver disease in that inhibition of p53 might possibly prevent steatotic hepatocytes from being damaged. Remarkably, it was recently demonstrated that inhibition of p53
protects liver tissue against endotoxin-induced hepatocellular
injury (20), increased sensitivity to which is reported to be
involved in the pathogenesis of fatty liver disease (37).
Acknowledgment—We thank Alyssa H. Hasty for critical reading of
the manuscript.
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