Supplemental Material can be found at:
http://www.jbc.org/cgi/content/full/M801824200/DC1
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 30, pp. 21220 –21229, July 25, 2008
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

Cyclin-dependent Kinase Inhibitor, p21WAF1/CIP1, Is Involvedin Adipocyte Differentiation and Hypertrophy, Linking toObesity, and Insulin Resistance*□
S

Received for publication, March 6, 2008 Published, JBC Papers in Press, April 29, 2008, DOI 10.1074/jbc.M801824200

Noriyuki Inoue‡, Naoya Yahagi§, Takashi Yamamoto‡, Mayumi Ishikawa‡, Kazuhisa Watanabe‡,
Takashi Matsuzaka§, Yoshimi Nakagawa‡, Yoshinori Takeuchi‡, Kazuto Kobayashi‡, Akimitsu Takahashi‡,
Hiroaki Suzuki‡, Alyssa H. Hasty¶, Hideo Toyoshima‡, Nobuhiro Yamada‡, and Hitoshi Shimano‡§1
From the ‡Department of Internal Medicine (Metabolism and Endocrinology) and §Graduate School of Comprehensive Human
Sciences and Center for Tsukuba Advanced Research Alliance, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8575,
Japan and ¶Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232

The process of adipocyte differentiation has been extensively
characterized in cultures of preadipocyte clonal cell lines suchas mouse 3T3-L1 and 3T3-F442A (1, 2). Adipogenesis requiresa sequence of events including growth arrest of proliferating
preadipocytes, coordinated reentry into the cell cycle with limited clonal expansion, and growth arrest before terminal differentiation during which lipid accumulation occurs. Thus, it isreasonable to assume that factors involved in cell cycle regulation may have important roles in the adipocyte differentiation* This research was supported in part by Grant-in-Aid for Scientific Researchand Scientific Research on Priority Areas, 2006, 1805200 from the Ministryof Education, Science, Sports, and Culture. The costs of publication of thisarticle were defrayed in part by 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.
□S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Figs. A1–A5 and Tables A1–A3.
1
To whom correspondence should be addressed. Tel.: 81-29-853-3053; Fax:
81-29-863-3074, E-mail: shimano-tky@umin.ac.jp.

21220 JOURNAL OF BIOLOGICAL CHEMISTRY

process. Cell cycle progression in mammals is governed by various complexes of cyclins and cyclin-dependent kinases(CDKs)2 as well as their inhibitors, of which p21 and p27 are themost widely studied. It was previously suggested that expression of p21 and p27 is changed during the adipogenesis (3). p21
has been reported to be induced by Foxo1 and has been implicated in entry of adipocytes into the clonal expansion phase of
adipogenesis; meanwhile, p27 is critical for adipocyte hyperplasia (3–5). The expression of both p21 and p27 is altered during
adipocyte differentiation; however, their precise role in adipogenesis, especially at the time of terminal differentiation,
remains unclear.
Obesity is caused by over-nutrition and decreased physicalactivity and is characterized by excess storage of lipids in adipose tissue, which can be accounted for by both adipocyte
hyperplasia and hypertrophy. Obesity often precedes insulin
resistance and precipitates type 2 diabetes and cardiovasculardiseases (6). Impaired adipocyte differentiation is also related toinsulin resistance. Based upon the potential roles of CDK inhibitors in cell cycle progression and apoptosis, it is reasonable tospeculate that CDK inhibitors could be involved in obesity andinsulin resistance through regulation of adipocyte differentiation and adipose tissue growth.
p21WAF1/CIP1 is the major CDK inhibitor and halts the cell
cycle at G1 (Refs. 7–10; for review, see Refs. 11 and 12). p21 is awell known target of p53, a stress response. In addition to causing G1 arrest, p21 protects cells from apoptosis (12). We
recently found that p21 is a direct gene target of SREBPs, crucialtranscription factors involved in lipogenesis and adipogenesis(13, 14). We also reported that p21 expression was highly upregulated in hypertrophic adipose tissue and in the liver inmodels of obesity (15, 16). These data prompted us to explore
the role of p21 in adipocyte differentiation and hypertrophy andto determine the involvement of p21 in obesity and insulin
resistance.

2

The abbreviations used are: CDK, cyclin-dependent kinase; RNAi, RNA-mediated interference; MEF, mouse embryonic fibroblast; HFHS, high fat highsucrose; PPAR, peroxisome proliferator-activated receptor; DEXA, dual
energy x-ray absorptiometry; TUNEL, terminal dUTP nick-end labeling;
C/EBP, CCAAT/enhancer-binding protein.

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Both adipocyte hyperplasia and hypertrophy are determinantfactors for adipocyte differentiation during the development ofobesity. p21WAF1/CIP1, a cyclin-dependent kinase inhibitor, is
induced during adipocyte differentiation; however, its precise
contribution to this process is unknown. Using both in vitro andin vivo systems, we show that p21 is crucial for maintaining adipocyte hypertrophy and obesity-induced insulin resistance. The
absence of p21 in 3T3-L1 fibroblasts by RNA-mediated interference knockdown or in embryonic fibroblasts from p21ⴚ/ⴚ mice
impaired adipocyte differentiation, resulting in smaller adipocytes. Despite normal adipose tissue mass on a normal diet,p21ⴚ/ⴚ mice fed high energy diets had reduced adipose tissue
mass and adipocyte size accompanied by a marked improvement in insulin sensitivity. Knockdown of p21 in enlarged epididymal fat of diet-induced obese mice and also in fully differentiated 3T3-L1 adipocytes caused vigorous apoptosis byactivating p53. Thus, p21 is involved in both adipocyte differentiation and in protecting hypertrophied adipocytes against apoptosis. Via both of these mechanisms, p21 promotes adipose
tissue expansion during high fat diet feeding, leading toincreased downstream pathophysiological consequences suchas insulin resistance.

Roles of p21 in Adipocyte Hypertrophy
EXPERIMENTAL PROCEDURESJULY 25, 2008 • VOLUME 283 • NUMBER 30

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Cell Culture and Adipocyte Differentiation—3T3-L1 cells(ATCC) were maintained in high glucose Dulbecco’s modified
Eagle’s medium (Invitrogen) with 10% fetal calf serum in 5%
CO2. Cells were seeded at 2.5 ⫻ 105 cell/6-cm collagen-coateddishes (BD Biosciences) (day 4) and cultured to confluence (day2). After 2 days, cells were replaced with differentiation induction medium A (5 ␮g/ml insulin, 1 ␮M dexamethasone, and 0.5
mM isobutylmethylxanthine) (day 0). After 48 h cells werechanged to differentiation induction medium B (5 mg/ml insulin) (day 2). Medium was renewed every other day.
Preparation of Recombinant Adenovirus—cDNAs encoding
the full-length fragment of mouse p21Waf1/Cip1 were integrated
into adenoviral vectors. RNAi adenovirus vectors, RNAi-LacZ(LacZi), p21 RNAi-137 (137i), and p21 RNAi-275 (275i) wereconstructed with BLOCK-iTTM U6 RNAi Entry Vector kit and
BLOCK-iTTM Adenoviral RNAi Expression system (Invitrogen). Each adenoviral vector was propagated in 293A cells andpurified by cesium chloride density centrifugation (17, 18).
Oil Red O Stain—Cells were fixed with 10% formalin in phosphate-buffered saline for 10 min at 37 °C and stained with Oil
Red O for 60 min at room temperature. Cells were washed with
distilled water, and the retained dye was eluted by isopropanol(19).
Mice—This project was approved by The Animal Care Committee of University of Tsukuba and performed under its guidelines. The p21waf1/Cip1 knock-out mice (B6;129S2-Cdkn1atm1Tyj/J)
were originally purchased from CLEA Japan Inc. and The Jackson Laboratory, provided by Dr. Tyler Jacks (20) and were backcrossed onto the C57BL/6J background within our colony forfive or more generations. Mice were genotyped by PCR according to methods provided by The Jackson Laboratory. Mice weremaintained on a normal chow diet for a 14-h light/10-h darkcycle. For diet-induced obesity, the mice were fed on a high fat
high sucrose (HFHS) diet as previously described (21) at 10
weeks of age for 6 (or indicated) weeks. We performed weekly
collections of blood for metabolic analysis (measurement ofblood glucose, insulin, triglyceride, total cholesterol, and freefatty acid) and measured body weight. They were subjected to
intravenous glucose tolerance tests 4 weeks after the HFHS dietstarted, insulin tolerance tests 5 weeks after the diet started,dual energy x-ray absorptiometry (DEXA) analysis, and sacrifice at 6 weeks. Tissue samples were fixed in 10% formalin for
microscopy analysis and preserved in liquid N2 for the Northern blot analysis.
DEXA Analysis—PIXImus2 DEXA (GE Medical Systems
LUNAR) was used to measure lean body tissue and percent fat
mass (22).
Determination of Adipocyte Number and Size—Epididymal
fat pads from normal mice (⬃50 mg) and HFHS-fed mice(⬃150 mg) were fixed in osmium tetroxide-collidine buffer for
3 days. Adipocytes of diameter from 25 to 125 ␮m in normal
mice or from 25 to 250 ␮m in HFHS mice were collected byfiltration through 25-, 125-, and 250-␮m nylon screens. Sizeand number of adipocytes were measured with a Z-2 Coultercounter for normal mice and a Multisizer 3 Coulter counter for

HFHS mice (Beckman Coulter) as previously described(22–24).
Metabolic Studies—Glucose tolerance tests were performed on male mice fasted overnight (9 –12 h). Glucose
solutions were injected at 2 g/kg of body weight via tail vein.
Blood glucose and insulin were measured 0, 5, 15, 30, and 60
min after injection. Insulin tolerance tests were performedon male mice fasted for 3 h. Mice were intraperitoneallyinjected with human regular insulin at 0.5 units/kg or 0.75
units/kg of body weight (Humulin R 40 units/ml, Eli Lilly),
and blood glucose was measured 0 –120 min after injection
with ANTSENSE II (Horiba Ltd.). Plasma glucose, triglyceride, cholesterol, and non-esterfied fatty acids were determined with Glu ICII, TG IE, T-Cho IE, and non-esterfied
fatty acid IC kits from WAKO. Plasma insulin and leptin
were determined with the mouse insulin enzyme-linked
immunosorbent assay (ELISA) kit (TMB) (AKRIN-011T,
Shibayagi) and the mouse leptin ELISA kit (Morinaga Institute of Biological Science, Inc.), respectively.
Northern Blot Analysis—Total RNA was prepared from livers, white adipose tissue, brown adipose tissue, muscle, andcultured cells using TRIzol reagent (Invitrogen). For Northernblot analysis, equal aliquots of total RNA from 5– 6 mice andculture cells were pooled (5–10 ␮g of total RNA), denatured
with formaldehyde and formamide, subjected to electrophoresis in a 1% agarose gel, and transferred to Hybond N membranes (Amersham Biosciences) for hybridization. cDNA
probes were labeled with [␣-32P]dCTP (3,000 –10,000 cpm)
using RediprimeTM Random Prime Labeling System (Amersham Biosciences). The filters were hybridized with the radiolabeled probe in Rapid-hyb buffer (Amersham Biosciences) at
65 °C and washed in 0.1 ⫻ SSC, 0.1% SDS at 65 °C. Blots were
exposed to Kodak BIOMAX MS1 (Eastman Kodak Co.) andBAS 2500 with BAStation software (Fuji Photo Film).
Cell Proliferation Assay—For determination of cell proliferation, a 5-bromo-2⬘-deoxyuridine labeling and detection kit I
was used according to the manufacturer’s instructions (Roche
Applied Science).
Apoptosis Assay—For the assessment of apoptosis, the In Situ
Cell Death Detection kit (Roche Applied Science) was used.
The test principle of this kit was labeling DNA strand breaks byterminal deoxynucleotidyltransferase, which catalyzes thepolymerization of labeled nucleotides to free 3⬘-OH DNA ends
in a template-independent manner (TUNEL reaction).
Immunoblot Analysis—Cells were harvested in 1% Triton
X-100 buffer (25 mM Hepes (pH 7.9), 50 mM KCl, 5 mM
EDTA, 5 mM MgCl2, 1% Triton X-100, 1 mM dithiothreitol,and protease inhibitor (Roche Applied Science)). Protein
concentrations were determined using the BCA protein
assay kit (Pierce). Samples were fractionated on 8, 12, or 15%
SDS-PAGE followed by transfer to Immobilon Transfer
membranes (Millipore). Blots were subsequently incubatedin Tris-buffered saline-Tween overnight at 4 °C with 1:2000
anti-p21waf1, anti-p27Kip1, anti-C/EBP␣, anti-C/EBP␤, antip53 (Santa Cruz) Ser-15 phosphorylation of p53, Ser-20
phosphorylation of p53, Ser-46 phosphorylation of p53 (Cell
Signaling), and ␣-tubulin (Calbiochem). After incubation
with horseradish peroxidase-conjugated secondary antibod-

Roles of p21 in Adipocyte HypertrophyCA-2 0 1

2 3 4

5

6

7

p21
α-tubulin

B
non

LacZi

137i

275i

p21

LacZi

137i

275i

1011 optical particle unit) was
injected on each side of the epididymal fat pad (25).
Statistical Analysis—Data areexpressed as the mean ⫾ S.E. Statistical significance was assessed using
the Student’s t test. Data sets involving more than two groups areassessed by Dunnett’s test (SASInstitute Inc.).

RESULTS
Effects of the Acute Absence of p21
on
Adipocyte Differentiation inDEDay-2 0 1 2 3 4 5 6Day
0 1 2 3 4 5 6Vitro—3T3-L1 fibroblasts were differentiated into adipocytes, and p21
LacZi
expression was assessed. Consistent
LacZiwith previous works, there was a
137i
rapid induction of p21 protein at the
137itime of differentiation into adipocytes (Fig. 1A) (3). Expression of p21
275i
275i
was sustained at both the mRNA
PPARγ
p21
and protein levels during terminal
differentiation and during the subDay
0 1 2 3 4 5 6Day-2 0 1 2 3 4 5 6sequent hypertrophic phase of lipid
accumulation. To determine the
LacZi
effects of acute p21 deficiency,
LacZiknockdown of p21 using two differ137i
ent adenoviral vectors with RNAi
137i
(137i and 275i) before induction ofdifferentiation was performed (Fig.
275i
275i
1B). p21 deficiency caused a dosep27
Adipsindependent suppression of adipocyte
differentiation as assessed by OilDay
0 1 2 3 4 5 6-2 0 1 2 3 4 5 6
Day
Red O staining (Fig. 1C). The inhibiLacZition of p21 resulted in no changes in
p27 levels (Fig. 1D). However,LacZi
reduction in p21 levels was accom137i
137i
panied by reduced levels of C/EBP␣
and PPAR␥, transcription factors
275i
275i
involved in adipocyte differentiation, along with adipsin, a marker
C/EBPα (p42)
C/EBPβfor adipogenesis (Fig. 1E). However,
FIGURE 1. Inhibition of adipocyte differentiation in 3T3-L1 cells by knockdown of p21. A, changes of p21
protein in differentiating 3T3-L1 cells. Total cellular proteins were extracted from cells at the indicated days of C/EBP␤, known to function at andifferentiation and subjected to immunoblot analysis with antibodies against p21 and ␣-tubulin as a loading initial stage of adipogenesis wascontrol. B, efficacy of p21 knockdown in 3T3-L1 preadipocytes infected with p21RNAi adenoviruses. Expressionof p21 and p27 level is shown by immunoblotting 24 h after the infection of p21 RNAi adenovirus (137i or 275i) rather enhanced by p21 inhibition,or LacZi adenovirus (LacZi) as a negative control. Note that 137i exhibits nearly complete suppression with a suggesting that the absence of p21
higher efficiency of p21 knockdown than 275i. C, effect of p21 gene knockdown on adipocytes differentiation. might impair the later stage of adi3T3-L1 adipocytes were infected with adenoviruses at day 3. The Oil Red O staining of cells from day 6 postdifferentiation induction is shown. D and E, time course analysis of protein (D) and mRNA (E) expression in pocyte differentiation (Fig. 1E).
3T3-L1 cells after infection of p21 RNAi adenoviruses at day 3. Cellular total RNA (5 ␮g) and protein (80 ␮g) were
Effects of the Chronic Absence of
subjected to SDS-PAGE followed by immunoblot analysis for p21, p27, and C/EBP␣ (D) and to Northern blot p21 during Adipocyte Differentiaanalysis for PPAR␥, adipsin, and C/EBP␤ (E), respectively.
tion in Vitro—To determine theeffect of chronic absence of p21,
mouseembryonic
fibroblasts(MEFs) from p21⫺/⫺ mice wereies, blots were detected by ECL or ECL Advance Western
induced to differentiate into adipocytes (supplemental Fig. A1).
blotting detection kit (Amersham Biosciences).
In Vivo Adenovirus Injection into White Adipose Tis- Before differentiation, p21⫺/⫺ MEF cells exhibited an increased
sue—After 3 weeks of HFHS feeding, mice were anesthetized cell number and uptake of 5-bromo-2⬘-deoxyuridine as comby halothane before tissue dissection. The adenovirus (2.5 ⫻ pared with p21⫹/⫹ control cells, indicating enhanced cellp27

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Roles of p21 in Adipocyte Hypertrophy
Metabolic Effects of the Chronic
Absenceof p21 in Vivo—Consistent
∗
35
35
∗
with
the
results from cultured cells,
37
∗
30
∗ 30
adiposity of chow diet-fed p21⫺/⫺
35
25
25
mice was normal. Growth curves
33
were nearly identical between
20
20
∗
∗
p21⫺/⫺ and p21⫹/⫹ mice (supple31
15
15∗
mental Fig. A2A). Body weight, total
∗
29
10
10
∗
fat weight (by DEXA), and adipo27
55cyte size did not significantly∗
025
0changed with only a minimal trend
0
1
2
3
4
5
6
Total
Lean
%Fat
toward a decrease in p21⫺/⫺ miceTime (weeks)(supplemental Fig. A2, B and C, andC
D 35000
1.8
2
A3, A–C). There were no significant
1.6changes in plasma glucose, insulin,
30000
1
∗
1.4
lipids, or leptin levels (supplemental∗
25000
Table A1). Insulin sensitivity and
1.2
20000
0
secretion did not change in p21⫺/⫺1
p21(+/+) p21(-/-)
mice as assessed by glucose and
15000
0.8
insulin tolerance tests (supplemen0.6
10000
tal Fig. A2, D and E, and Table A1).
0.4
5000
Next, the role of p21 in adipogen0.2
esis in diet-induced obesity was
00p21(-/-)
evaluated. Our preliminary datap21(+/+)
25 50 75 100 125 150 175 200 225 250
diameter of adipocyte (µm)
indicated that gene expression ofE
p21(+/+)
p21(-/-)
p21 was highly induced by high fat
diet, suggesting a role of this proteinin the hypertrophic change of adipocytes (data not shown). Whenp21⫺/⫺ mice were fed the HFHS
diet, which rapidly induces obesity
and insulin resistance, the degree ofbody weight gain was ameliorated
compared with strain-matched
C57BL/6 p21⫹/⫹ controls (Fig. 2A).
F
The retarded body weight gain in
p21⫺/⫺ mice was due to decreased
fat mass as estimated by DEXA
(Fig. 2B). The weight of epididymal
fat pads was also significantly
decreased in p21⫺/⫺ mice (Fig. 2C).
Total fat weight of p21⫹/⫺ mice was
between those of p21⫹/⫹ andp21⫺/⫺ mice, demonstrating the
FIGURE 2. Prevention of diet-induced obesity in p21ⴚ/ⴚ mice. p21⫹/⫹, p21⫹/⫺, and p21⫺/⫺ mice wereplaced on a high fat and high sucrose (HFHS) diet at 10 weeks of age for a total of 6 weeks. A, body weight curves dose-dependent effect of p21 on
on a HFHS diet. B, total and lean mass and % fat in p21⫹/⫹ (white bars), p21⫹/⫺ (gray bars), and p21⫺/⫺ (black adiposity (Fig. 2B). The size distribars) mice as estimated by DEXA. C, weight of epididymal fat pads in p21⫹/⫹ and p21⫺/⫺ mice. WAT, white bution of adipocytes from the epiadipose tissue. D, size distribution and total cell number (inset) of adipocytes from white adipose tissue. E andF, microscopic examination of white (E) and brown (F) adipose tissues (hematoxylin/eosin stain). Values repre- didymal adipose tissue, as estisent the means ⫾ S.E. from 14 mice per group. p ⬍ 0.05 (*) and at p ⬍ 0.01 (**) for p21⫺/⫺ compared with mated by a Coulter counter, alsop21⫹/⫹ groups.
confirmed a significant decreasein the size of p21⫺/⫺ adipocytes
growth in the absence of p21 (supplemental Fig. A1, A and B). (Fig. 2D). Consistently, histological analysis of adipose tissue
Upon induction of differentiation, p21⫺/⫺ MEF cells began to demonstrated that the marked enlargement of adipocytes indifferentiate sooner; however, at terminal differentiation the HFHS-fed mice was suppressed in p21⫺/⫺ mice (Fig. 2E).
size of the resultant adipocytes was smaller than differentiated The presence of large lipid vacuoles, which emerged in thewild type p21⫹/⫹ MEFs (supplemental Fig. A1C). These data cytosol of brown fat cells of HFHS diet-fed p21⫹/⫹ mice, wasdemonstrate that p21 contributes to, but is not indispensa- decreased in p21⫺/⫺ mice (Fig. 2F). In fact, brown adipose
ble, for adipocyte differentiation.
tissue of p21⫺/⫺ mice fed HFHS diets appeared similar to
A

B

40

40

Weight (g)Cell number

Total cell number (×106 )JOURNAL OF BIOLOGICAL CHEMISTRY

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JULY 25, 2008 • VOLUME 283 • NUMBER 30

Fat (%)WAT Weight (g)Body Weight (g)39Roles of p21 in Adipocyte Hypertrophy

21224 JOURNAL OF BIOLOGICAL CHEMISTRY

A

600

Glucose (mg/dL)

500
400
300
200
100
0

0

5

15

∗

∗

5

15

30
Time (min)60

30Time (min)

60

3000

Insulin (pg/mL)

2500
2000
1500
1000
500
0

B

0

200

Glucose (mg/dL)

180
160
140
120
100

∗
∗

80
60

∗
∗

∗
∗

40
0

15

30

60
Time (min)

∗
∗
90

∗∗
120

FIGURE 3. Protection of p21ⴚ/ⴚ mice from insulin resistance caused by dietinduced obesity. Glucose tolerance test (A) and insulin tolerance test (B) on
p21⫹/⫹ (white circles) and p21⫺/⫺ mice (black circles). Mice were fed the HFHS dietfor 4 weeks and injected with 2 mg/kg D-glucose (A) or 0.75 units/kg insulin for 5
weeks under the HFHS diet condition (B). The panels show serum glucose concentration (upper panel of A and B) and insulin concentration (lower panel of A) at
the indicated time (min). Values represent the means ⫾ S.E. from 14 mice pergroup. p ⬍ 0.05 (*) and at p ⬍ 0.01 (**) for p21⫺/⫺ compared with p21⫹/⫹ groups.

adipocytes could induce apoptosis, suppressing the hypertrophic changes required for completion of differentiation. p53,
the most potent inducer of p21, is known to be activated in
VOLUME 283 • NUMBER 30 • JULY 25, 2008

Downloaded from www.jbc.org at University of Tokyo Library on October 1, 2008p21⫹/⫹ mice on a chow diet (compare Fig. 2F and supplemental Fig. A3D).
The amelioration of obesity was accompanied by marked
improvement of insulin resistance (Fig. 3). Improved insulin
sensitivity in HFHS-fed p21⫺/⫺ mice was supported bydecreased plasma insulin levels despite similar or slight trendsof decreasing plasma glucose levels in fasted and fed conditions
and in intravenous glucose tolerance tests (supplemental Table
A2, Fig. 3, A and B). Insulin tolerance tests showed a more
prominent decrease in plasma glucose levels after injection of
insulin in p21⫺/⫺ mice than in control mice (Fig. 3B). These
data indicate that gene disruption of p21 causes a partial protection from diet-induced obesity and a marked improvement
in insulin resistance. This protection from diet-induced obesity
was not due to differences in food intake (2.95 ⫾ 0.10 versus
2.86 ⫾ 0.05 g/day for p21⫹/⫹ and p21⫺/⫺ mice, respectively;n ⫽ 6; data were collected over a 4-week period).
Gene Expression in Metabolic Tissues of p21⫺/⫺ Mice—Expression of various genes in white adipose tissue of HFHS-fed
p21⫹/⫹ and p21⫺/⫺ mice were estimated by Northern blot
analysis (Fig. 4A). Adipogenic transcription factors such as
PPAR␥, C/EBP␣, and C/EBP␤ were not different between
p21⫹/⫹ and p21⫺/⫺ mice. Expression of leptin and ADRP,
known to be up-regulated in white adipose tissue in obesity, wassuppressed in p21⫺/⫺ mice on a HFHS diet, whereas adipsin,
known to be suppressed in obesity, was up-regulated in p21⫺/⫺
adipose tissues to varying degrees. As estimated by reverse transcription-PCR (supplemental Fig.A5A), decreased leptin andincreased adiponectin were observed, consistent with amelioration of obesity in p21⫺/⫺ mice on a HFHS diet (supplemental
Fig. A5A). Expression of monocyte chemoattractant protein 1
and tumor necrosis factor ␣ was also decreased, indicating thatmacrophage infiltration could be suppressed by the absence ofp21 (supplemental Fig. A5B). PGC1␣ in brown adipose tissue,
SCD-1 in the liver, and PPAR␦ in skeletal muscle are all known
to be involved in energy consumption. These genes weredecreased in diet-induced obesity in p21⫹/⫹ mice, and theirlevels were normalized in p21⫺/⫺ mice, indicating a shift ofenergy balance toward to expenditure (Fig. 4B).
Potential Involvement of Apoptosis in Mechanisms by Which
p21 Deficiency Protects against Diet-induced Obesity—To
determine the mechanism by which the absence of p21 protects
against diet-induced obesity and insulin resistance, p21 was
suppressed in 3T3-L1 cells after they had already differentiatedinto mature adipocytes. In contrast to infection of adenoviral
RNAi before the differentiation protocol (Fig. 1, C–E), acutesuppression of p21 at day 6 caused inhibition of lipid accumulation, detachment of the lipid-containing cells, and a marked
change in color of the medium (Fig. 5A). TUNEL staining confirmed that these changes significantly involve apoptosis (Fig. 5,
B and C). Supporting these observations, immunoblot analysis
indicated that inhibition of p21 was followed by a time-dependent induction of caspase-3, the terminal player of apoptosis, but
no change in expression of Bcl-2, a gene that plays a protectiverole from apoptosis (Fig. 6B). p53 phosphorylated at Ser-46,
associated with apoptosis (26), was induced at day 8, 48 h after
infection with p21RNAi adenovirus (Fig. 6B). These data suggest that p21 inhibition during the terminal differentiation ofRoles of p21 in Adipocyte Hypertrophy
A

A

HFHS

NC HFHS+/+ +/++/+ +/--/-

p21

MediumPPARγ
C/EBPαC/EBPβOil Red O

Adipsin

ADRP

B

LacZi

137i

275i

Phase

HoechstTUNEL

Downloaded from www.jbc.org at University of Tokyo Library on October 1, 2008

LeptinLacZiAdiponectin
Resistin
137i

36B4

B

NC
+/+

+/-

-/-

+/+

+/-

-/-

36B4

BAT

275i

SCD1

PGC-1α36B4Muscle PPARδ
36B4
FIGURE 4. Gene expression of p21ⴚ/ⴚ mice on a HFHS diet. A, Northern blot
analysis in epididymal fat pads in p21⫹/⫹ and p21⫺/⫺ mice fed a HFHS diet for
6 weeks. Left panel, p21⫹/⫹ mice on a normal chow (NC) diet as a control and
HFHS diet. Right panel, p21⫹/⫹, p21⫹/⫺, and p21⫺/⫺ mice on a HFHS diet for 6
weeks. B, Northern blot analysis of livers, blown adipose tissues (BAT) andskeletal muscles in p21 knock-out mice was shown. Left panel, p21⫹/⫹,p21⫹/⫺, and p21⫺/⫺ mice on a normal chow diet as a control. Right panel,p21⫹/⫹, p21⫹/⫺, and p21⫺/⫺ mice fed a HFHS diet for 6 weeks. All mice weresacrificed at fasted state for 12 h. Total RNA was extracted from the organs ofsix mice. Equal aliquots of the RNA samples were pooled for each organ (10␮g) and subjected to Northern blot analysis for hybridization with the indicated cDNA probes (36B4 as a loading control).

response to cellular stresses. p53 protein has been detected in
3T3-L1 cells and remains constant during intracellular accumulation of lipids in Fig. 6A, consistent with the previous report(27, 28). However, phosphorylation of p53 protein at Ser-15 andSer-20 is the key process for p21 trans-activation (29, 30). In
accordance with p21 induction, the phosphorylation of thesesites was elevated during the adipocyte differentiation. In
JULY 25, 2008 • VOLUME 283 • NUMBER 30

C

6
TUNEL positive cells (%)

Liver

HFHS

∗∗∗

5
4

∗∗∗

3
2
1
0
LacZi

137i

275i

FIGURE 5. p21 gene knockdown induces apoptosis in 3T3-L1 adipocytes.
A, 3T3-L1 cells were differentiated into adipocytes, and p21 RNAi adenovirus
was infected at day 6. Media (upper panel) and Oil Red O staining of 3T3-L1
cells (lower panel) 4 days after infection of p21 RNAi (137i or 275i), or control
LacZi adenovirus (day 10). B, apoptosis of 3T3-L1 adipocytes after p21 knockdown at day 10. Microscopic examinations of the cells with light phase contrast for morphology (phase (left)), Hoechst 33342 for nuclear staining(Hoechst (middle)), and TUNEL staining for apoptotic cells (TUNEL (right)) are
shown at a magnification of ⫻400. C, emergence of apoptotic cells was determined by the ratio of TUNEL positive cells to Hoechst 33342-positive cells.
Values represent the mean ⫾ S.E. from six dishes per group. ***, at p ⬍ 0.0001
for 137i and 275i versus LacZi adenovirus-infected cells.

contrast, phosphorylation of p53 at Ser-46 was reported to
be associated with apoptosis (26). This apoptosis-relatedchange of p53 was induced at the late stage of differentiationin the 3T3-L1 adipocytes after the Ser-15 and Ser-20 phosphorylation (Fig. 6A).
JOURNAL OF BIOLOGICAL CHEMISTRY

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Roles of p21 in Adipocyte Hypertrophy
A

Day

-2

0

1

2

3

4

5

6

p21
p53
P-Ser15 p53
P-Ser20 p53
P-Ser46 p53
C/EBPαα-tubulin

co

Day10

Day12

nt
.
La
cZ
i
27
5i
13
7i
co
nt
.
La
cZ
i
27
5i
13
7i

Day8
nt
.
La
cZ
i
27
5i
13
7i

Day6

co

B
p21

P-Ser46 p53
Caspase-3
Bcl-2
α-tubulin

FIGURE 6. Activation of caspase-3 by p21 knockdown in 3T3-L1 adipocytes after intrinsic activation of p21/p53. A, time-course changes of p53
and p21 activation in 3T3-L1 cells during adipocyte differentiation. 3T3-L1
fibroblasts were differentiated into adipocytes according to the standard protocol (see “Experimental Procedures”). The level of p53, p21 proteins, andphosphorylation (P) of p53 at Ser-15, Ser-20, and Ser-46 crucial for p21 transactivation were analyzed at the indicated days by immunoblot analysis.
B, 3T3-L1 cells were induced to differentiate into adipocytes and infected with
p21 RNAi adenovirus at day 6. Changes of p21, p53, Ser-46 phosphorylation of
p53, caspase-3, Bcl-2, and ␣-tubulin (as a loading control) proteins in 3T3-L1
adipocytes at the indicated days after the induction for adipocyte differentiation were determined by immunoblot analysis.

To examine the involvement of apoptosis in the suppression of p21 in vivo, adenoviral RNAi for p21 was injected
directly into epididymal adipose tissue of HFHS-fed mice.
Significant inhibition of p21 expression was obtained 4 days
after the infection (Fig. 7A), causing a trend to loss of bodyweight (Fig. 7B) accompanied by decreased glucose and insulin levels in insulin and glucose tolerance tests, respectively(Fig. 7, C and D). TUNEL staining demonstrated induction of
adipocyte apoptosis by p21 knockdown (Fig. 7E). These data
implicated that p21 could be induced by diet-induced obesity and contributes to protection from apoptosis during adipocyte hypertrophy.

DISCUSSION
Role of p21 in Adipocyte Differentiation and Apoptosis—
Our current study clearly demonstrates that p21 plays a crucial role in later stages of differentiation and hypertrophy of
adipocytes. p21 is induced during adipogenesis and sustained during terminal differentiation. p21 knockdown
experiments demonstrated that p21 contributes to but is not
absolutely required for adipocyte differentiation. Emergenceof vigorous apoptosis upon acute p21 knockdown during theterminal phase of 3T3-L1 differentiation and in hypertrophic adipose tissue during diet-induced obesity in mice
reveals that p21 is crucial for adipocyte hypertrophy rather

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p53

than for the initial stages of differentiation. In vivo, p21 washighly induced in enlarged adipose tissue of diet-induced
obese mice, and its absence ameliorated obesity. In contrast
to acute knockdown experiments of p21, apoptosis was notdetected in adipose tissue from p21⫺/⫺ mice on a HFHS dietor in p21⫺/⫺ MEF throughout adipocyte differentiation(data not shown). We speculate that in the absence of p21,
adipocytes might not become hypertrophic enough to cause
apoptosis.
In normal physiology, it is conceivable that the increased
accumulation of intracellular lipids, which occurs during differentiation, is a cellular stress to large adipocytes, leading to activation of the p53/p21 pathway. In this situation p21 might berequired for survival of hypertrophic adipocytes. This potentialof p21 is mediated through its anti-apoptotic actions rather
than its growth arrest properties. It has long been speculated
that adipocyte hypertrophy involves apoptosis (31, 32), but evidence of apoptosis was not appreciably observed. Our data provide evidence that in the presence of p21, hypertrophic adipocytes are relatively protected against apoptosis, possibly
explaining this discrepancy.
Role of p53 in Sustained p21 Expression in Adipocytes—
Previous studies have shown that the transcription factors
Foxo1, SREBP-1c/ADD1, and C/EBP family are involved inadipocyte differentiation, and all three have strong transcriptional activity for the p21 promoter (4, 13, 33, 34). Eachof these factors likely contributes to the induction of p21
during the initial stages of differentiation in normal adipocytes. Although expression of p53, a dominant regulator of
p21, remains stable at both mRNA and protein levels during
adipocyte differentiation, we found that Ser-15 and Ser-20 of
p53, crucial sites for trans-activation of p21 (29, 35) were
hyperphosphorylated during the later stages of differentiation. It can be speculated that sustained p21 expression during terminal differentiation or obesity-related activation ismediated through p53 activation. We previously observedthat the p53/p21 pathway is activated in hypertrophied adipocytes, such as from leptin-deficient ob/ob mice, and also
in fatty livers (15, 16). This is consistent with our current
observation in adipose tissue of HFHS diet-induced obese
mice. p53 is the key sensor of indications of cellular stresssuch as DNA damage and ultimately determines whether thecell will go through cell growth arrest or apoptosis. p21 is anestablished cell cycle arrest inducer but has been also implicated to be involved in inhibition of apoptosis (12). In 3T3L1
adipocytes, p53 activation, as indicated by phosphorylation
at Ser-15 and Ser-20, could result from lipid accumulation.
Furthermore, phosphorylation of p53 at Ser-46 at the laterstage of differentiation indicated the potential risk of apoptosis (26) that was masked by p21. Our current data suggestthat hypertrophic adipocytes in obesity are also under considerable stress and are vulnerable to apoptosis, which undernormal conditions, could be inhibited by activation of p21.
Thus, we postulate that p21 plays a role in adipogenesis byhalting cell cycle but, more importantly, by preventing the
apoptosis of hypertrophic adipocytes.
Effect of p21 on Obesity and Insulin Resistance—Of clinicalrelevance, amelioration of high fat diet-induced obesity inRoles of p21 in Adipocyte Hypertrophy
we speculate that it could be strain
and/or sex differences in which weused male p21⫺/⫺ mice backcrossed
110
p21
to C57BL6 versus the original work
on female mice on the undescribed∗100back ground. We presume that p21
Leptin90∗∗∗
in adipocytes plays the major role inobesity and insulin resistance since80
even partial knockdown of p21 in
36B4
epididymal fat pads effectively
70
LacZi
137i
275i
reproduced the apoptosis anddecreased the size of adipocytes as
B 27.0D
900
observed in p21⫺/⫺ mice on HFHS
800
diet. Because decreased size of adi26.5
pose tissue was associated with
700
reduction in monocyte chemoat600
tractant protein 1 and tumor
26.0500
necrosis factor ␣ expression (sup400
plemental Fig. A5B), macrophage
25.5
infiltration and subsequent pro300
inflammatory responses in obesity
200
25.0
0 5
15
30
60
were suppressed in p21⫺/⫺ mice.
Time (min)
LacZi
137i
275i
The absence of p21 in macrophages
could contribute to inhibition ofE
Phase
Hoechst
TUNEL
inflammation in adipose tissues asreported in atherosclerosis (36).
There were only mild changes in theexpression of genes related to
LacZienergy metabolism in liver and skeletal muscle of p21⫺/⫺ mice on
HFHS diet, indicating that they are
not the primary organs responsible
for anti-obesity in the absence of
p21. It should be noted that in
137i
brown adipose tissue, PGC1␣ wasslightly restored in p21⫺/⫺ mice.
Diet-induced obesity is usuallyaccompanied by fat accumulationand decreased burning function inthis tissue (6), which was prevented
in HFHS-fed p21⫺/⫺ mice Consid275i
ering the similar food intake
between p21⫹/⫹ and p21⫺/⫺ mice,
it is possible that p21 deficiency
FIGURE 7. Local p21 knockdown in white adipose tissue from diet-induced obesity mice causes apopto- contributed to amelioration of dietsis. C57BL/6J mice (6 weeks of age) were fed a HFHS diet for 3 weeks. Adenoviral p21 RNAi (137i (black) or 275i induced obesity and insulin resist(gray)) or LacZi (white) as a control was directly injected into epididymal white fat pads. After injection of
adenovirus, these mice were fed a HFHS diet. Four days after the local infection, mice were sacrificed, and ance at least partially by restoring
adipose tissues were collected. Total RNA of equally pooled aliquots (10 ␮g) extracted from the fat pads of six the fat-burning function of brown
mice was subjected to Northern blot analysis for p21 and leptin (A). Body weights (BW), blood glucose concen- fat tissue.
trations 5 min after intraperitoneal injection of insulin 4 days after the local infection (C), and plasma insulin
A New Link between Cell Growthconcentrations on an intravenous glucose tolerance test 6 days after the local infection (D) are shown in the
diet-induced obesity mice after p21 knockdown in white adipose tissue (n ⫽ 12). Shown are TUNEL assays for and Metabolism—Our study proapoptotic cells with light phase contrast microscopy and Hoechst 33342 staining for nuclear staining 4 days
after the local infection (E). Values represent the means ⫾ S.E. from 12 mice per group. p ⬍ 0.05 (*) and at p ⬍ vides evidence that factors responsible for the regulation of cell0.001 (***) for 137i and 275i versus LacZi adenovirus infected mice.
growth and survival are alsop21⫺/⫺ mice was associated with improved insulin sensitivity. involved in metabolism. A recent report on a direct interacAdiposity in p21⫺/⫺ mice was originally reported to be slightly tion between p21 and Akt substantiates this novel linkincreased as estimated by epididymal fat pads (22). The cause of between a CDK inhibitor and metabolism (37). The p53/p21
this discrepancy with our data is currently unknown; however, pathway might play a determining role in the fate of hyper-

A

137i

275i

C

120

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Insulin (pg/mL)

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BW (g)

Glucose (mg/dL)

LacZi

Roles of p21 in Adipocyte Hypertrophy

Acknowledgments—We are grateful to Drs. Hiroshi Sakaue andTakehiro Nakamura for analysis of adipocyte cell size by Coultercounter. We thank Yuko Tamai for management of mice.
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trophic adipocytes in situations of excess energy. In the context of obesity-related diseases including insulin resistanceand diabetes, inhibition of p21 in adipocytes might have atherapeutic effect by suppressing adipose tissue expansion.
However, it is also possible p21-mediated adipocyte differentiation is important for proper storage of excess lipids inadipose tissue, thereby protecting against ectopic lipid storage and insulin resistance. Further studies are needed toclarify the precise mechanism by which p53/p21 pathway isregulated and involved in obesity and insulin resistance inadipose tissue. Recent reports on another CDK inhibitor,
p27, as well as Skp2, the F-box protein that controls ubiquitin-mediated degradation of p27, implicates the Skp2/p27
pathway in determining the cell number of adipocytes andpancreatic ␤ cells (5, 22, 38 – 40). Mice that lack CDK4
develop insulin-dependent diabetes as a result of a reduction
in islet mass, whereas deletion of p27 ameliorates hyperglycemia in diabetic mice by maintaining compensatory islet
hyperplasia and hyperinsulinemia, suggesting that these factors are involved in cell cycle regulation and could also play arole in ␤ cell mass and function (38, 41). However, p21 did
not have a significant impact on ␤ cell function in our study(supplemental Table A3) as recently reported (42). Taken
together, these data implicate p21 in controlling adipocyte
cell size and p27 in determining cell number, reflecting distinct regulations and adipose tissue growth by both of the
major CDK inhibitors.
In conclusion, our data demonstrate that p21 plays dual
roles in both adipocyte differentiation through cell cycle
arrest and in adipocyte hypertrophy through its anti-apoptotic action. Via these mechanisms mutually connected, p21
supports adipose tissue expansion during obesity linking toinsulin resistance. Our data suggest that a cell cycle regulator
can be involved in a wide range of physiology and pathophysiology in adipocytes, providing a new link between cell
growth and metabolism.

Roles of p21 in Adipocyte Hypertrophy
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