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
Received for publication, March 6, 2003 , and in revised form, April 14, 2003.
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ABSTRACT |
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INTRODUCTION |
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In the insulin signaling pathways, a transcription factor sterol regulatory element-binding protein-1 (SREBP-1) has recently been established to be a key molecule for the transcriptional regulation of triglyceride synthesis (10). SREBPs are members of the basic helix-loop-helix leucine zipper family of transcription factors that regulate fatty acid and cholesterol synthesis (reviewed in Refs. 1113). Whereas SREBP-2 plays a crucial role in regulation of cholesterol synthesis, SREBP-1 controls the transcription and expression of lipogenic enzymes such as fatty acid synthase (FAS) (reviewed in Refs. 1417). In fact, SREBP-1 and its downstream lipogenic enzymes are drastically induced when fasted animals are refed (18). These lipogenic genes belong to the group of genes that are induced most strongly by glucose/insulin2 and can be regarded as indicators of insulin signaling.
We have recently reported that the refeeding responses of SREBP-1 and its downstream lipogenic enzymes are markedly suppressed in adipocytes of ob/ob mice, which is presumably associated with impaired insulin signaling (19). Although the precise role of this down-regulation is currently undefined, it could be a negative feedback mechanism to prevent excess fat accumulation in extremely obese animals.
The p53 gene was the first tumor suppressor gene to be identified and has
been found to be inactivated in most human cancers
(20). The p53 protein is
responsible for preventing division of stressed cells and even causes
programmed cell death (apoptosis) through activation and/or suppression of the
transcription of target genes. For example, -irradiation activates p53
to turn on the transcription of p21Waf1/CIP1, which binds to and
inhibits cyclin-dependent kinases, thus blocking the G1-S and
G2-to-mitosis transitions. p53 not only activates transcription of
genes such as p21 through its response element, but also represses genes
lacking the element by binding to and sequestering essential transcription
factors such as TATA-binding protein
(21,
22). The stresses that
activate p53 are diverse, ranging from DNA damage to oxidative stress,
hypoxia, and heat shock (23).
The cytostatic and cytotoxic effects of TNF
were also demonstrated to
be mediated, at least in part, by p53 activation
(2426).
Thus, p53 has been thought to be a guardian angel against cellular stresses.
Especially, it has been extensively studied and well established as a tumor
suppressor. However, other roles of p53 beyond tumor suppression are still
obscure.
Considering that TNF is relevant to both cell growth and metabolic
events and that its effects are partly mediated by p53, we speculated that p53
could be involved in situations of metabolic deterioration associated with
insulin resistance. It is possible that p53 as a general repressor of gene
transcription could prevent insulin-responsive genes from being activated.
Moreover, a previous report on the gene expression profile of
ob/ob mouse adipose tissue examined by DNA microarray
analysis has revealed that p21 and Bax
, both of which are well-known
p53 target genes, are increased from 2- to 3-fold in ob/ob
mice (27).
Based on these facts, we hypothesized that hypertrophied adipocytes are under various stresses that induce p53, which in turn suppresses lipogenesis in a negative feedback regulation. In our present study, we discovered that p53 is induced upon refeeding in ob/ob adipocytes and activates its target genes including p21. In addition, p53 is involved in the suppression of SREBP-1 and the concomitant down-regulation of lipogenic enzymes.
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EXPERIMENTAL PROCEDURES |
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Nuclear Protein Extraction and ImmunoblottingNuclear
extract protein from white adipose tissue was prepared as described previously
(27). Briefly, fresh adipose
tissue (3 g pooled from 310 male mice) was rinsed in ice-cold PBS,
minced, and homogenized with 10 strokes of a Teflon homogenizer in 15 ml of
NDS buffer at 4 °C (10 mM Tris, pH 7.5, 10 mM NaCl,
60 mM KCl, 0.15 mM spermine, 0.5 mM
spermidine, 14 mM mercaptoethanol, 0.5 mM EGTA, 2
mM EDTA, 0.5% Nonidet P-40, 1 mM dithiothreitol)
supplemented with protease inhibitors (12.5 µg/ml
N-Acetyl-Leu-Leu-norleucinal-CHO (ALLN, Calbiochem), 2.5 µg/ml
leupeptin, 2.5 µg/ml aprotinin, 2.5 µg/ml pepstatin A, 0.1 mM
phenylmethylsulfonyl fluoride). Nonidet P-40 concentration was increased to 1%
and nuclei were pelleted at 700 x g for 10 min, washed once
with 25 ml NDS buffer (1% Nonidet P-40), filtered through 70 µm mesh,
pelleted at 500 x g for 10 min, resuspended in 1 volume of 1%
citric acid, lysed by the addition of 2.5 volumes of 0.1 M Tris,
2.5% SDS and 0.1 M dithiothreitol, sonicated briefly, and heated to
90 °C for 5 min. Aliquots of nuclear protein (15 µg) were subjected to
SDS/PAGE. p53 was detected 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-p53Ser-15 (9284S, New England Biolabs). To
confirm equal loading of nuclear protein, a 1:500 dilution of anti-c-myc
(sc-764, Santa Cruz Biotechnology) was also used. Bound antibodies were
detected using a horseradish peroxidase-coupled anti-rabbit IgG secondary
antibody (Amersham Biosciences) and visualized using ECL Western blotting
detection system kit (Amersham Biosciences).
Northern BlottingTotal RNA from epididymal fat pad was
extracted using Trizol reagent (Invitrogen), and 10 µg RNA samples equally
pooled from each group (n = 36) were run on a 1% agarose gel
containing formaldehyde and transferred to a nylon membrane. For experiments
with isolated adipocytes, total RNA from adipocytes isolated by collagenase
digestion was prepared as previously described
(29). In brief, fresh adipose
tissue (4 g) was minced in 8 ml buffer A (10 mM HEPES at pH
7.4, 130 mM NaCl, 5.2 mM KCl, 1.3 mM
KH2PO4, 2.7 mM CaCl2, 1.3
mM MgSO4, 25 mM NaHCO3, 3% BSA, 2
mM glucose, 200 nM adenosine) at 37 °C and mixed
with equal volume of buffer A containing 4 mg/ml collagenase (type II for
adipocyte, Sigma). Following 1-hour digestion with gentle shaking at 37
°C, adipocytes were filtered through 250 µm nylon mesh, washed with 30
ml of buffer A, transferred to another tube, and mixed with 4 volumes of
Trizol reagent (Invitrogen). Total RNA sample (10 µg) from one mouse was
applied on each lane. The cDNA probes for SREBP-1, fatty acid synthase, ATP
citrate lyase, spot 14 and 36B4 (acidic ribosomal phosphoprotein P0) were
cloned as described previously
(30,
31). The cDNA probes for p53,
p21, p27, mdm-2, Bax
, insulin-like growth factor-binding protein-3
(IGFBP-3), glycerol-3-phosphate dehydrogenase (GPDH) and tumor necrosis factor
(TNF
) were prepared by cloning RT-PCR products from mouse liver
or adipose tissue RNA into pGEM-T easy vectors (Promega). The primers used for
PCR were as follows: for p53, 5' primer was
5'-GGAAATTTGTATCCCGAGTATCTG-3' and 3' primer was
5'-GTCTTCCAGTGTGATGATGGTAA-3'; for p21, 5' primer was
5'-TGTCCAATCCTGGTGATGTC-3' and 3' primer was
5'-TCTCTTGCAGAAGACCAATCTG-3'; for p27, 5' primer was
5'-GTCAAACGTGAGAGTGTCTAACG-3' and 3' primer was
5'-GCGAAGAAGAATCTTCTGCA-3'; for mdm-2, 5' primer was
5'-CCAGGCCAATGTGCAATAC-3' and 3' primer was
5'-GTGAGCAGGTCAGCTAGTTGAA-3'; for Bax
, 5' primer was
5'-GCTCTGAACAGATCATGAAGACA-3' and 3' primer was
5'-CATGATGGTTCTGATCAGCTC-3'; for IGFBP-3, 5' primer was
5'-CATGCCAAGATGGATGTCATC-3' and 3' primer was
5'-GAGGCAATGTACGTCGTCTTTC-3'; for GPDH, 5' primer was
5'-CCATGGCTGGCAAGAAAGT-3' and 3' primer was
5'-AATCACTTCAGAAATGAGCTTCAG-3'; for TNF
, 5' primer
was 5'-GGTTCTGTCCCTTTCACTCACTG-3' and 3' primer was
5'-TTGACCTCAGCGCTGAGTTG-3'. 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
and TNF
, for which ULTRAhyb hybridization buffer (Ambion) was used at
42 °C. The membranes were washed in 0.1x SSC, 0.1% SDS at 65 °C.
Blots were exposed to Kodak XAR-5 film.
Plasmid ConstructionsLuciferase gene constructs containing 2.6-kb fragments of the mouse SREBP-1c promoter (pBP1c-Luc) and 0.24-kb fragments of the rat fatty acid synthase promoter (pFAS-Luc) were prepared as described previously (32, 33). Plasmid pGPDH-Luc was constructed by PCR amplification of 489 bp to +23 bp of the mouse glycerol-3-phosphate dehydrogenase (GPDH) promoter region (34) and insertion of the PCR products into pGL2-basic vector (Promega). Plasmid p53-Luc, a luciferase reporter plasmid containing 14 tandem copies of p53-binding motif (TGCCTGGACTTGCCTGG) and the luciferase gene was purchased from Stratagene. Mouse p53 expression plasmid driven by the CMV promoter (pCMV-p53) was constructed by inserting a DNA fragment amplified with PCR using first strand cDNA from mouse adipose tissue as a template, two primers (5' primer: 5'-TGGCTGTAGGTAGCGACTACAGTTA-3' and 3' primer: 5'-AGGCAGTCAGTCTGAGTCAGG-3') and Platinum Pfx DNA polymerase (Invitrogen) into pCMV7 vector (35). All DNA fragments generated with PCR were verified by sequencing.
Transfections and Luciferase Assaysp53-null human
osteogenic sarcoma cell line SaOS-2 was obtained from the American Type
Culture Collection (ATCC). SaOS-2 cells were cultured in Dulbecco's modified
Eagle's medium containing 25 mM glucose, 100 units/ml penicillin,
and 100 µg/ml streptomycin sulfate supplemented with 10% fetal calf serum
under 5% CO2 at 37 °C. On day 0, cells were plated on a 12 well
plate at 5 x 104 cells/well. On day 3, pCMV-p53 plasmid (1.0
µg), each luciferase reporter plasmid (0.25 µg), and a
CMV--galactosidase reference plasmid (pCMV-
-gal, Promega, 0.1
µg) were transfected into SaOS-2 cells using FuGENE 6 transfection reagent
(Roche Applied Science) according to the manufacturer's protocol. As a
control, the same amount of empty vector pCMV7 was transfected instead of
pCMV-p53 plasmid. After 48-h incubation, cells were harvested, and the amount
of luciferase activity in transfectants was measured by standard kits
(Promega).
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RESULTS |
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Next we evaluated the mRNA abundance of p53 under various nutritional conditions. To our surprise, the increased expression of p53 in ob/ob mouse adipose tissue was limited to a fed state and no difference was observed in a fasted state (Fig. 2a). The mRNA elevation of p53 appeared to be fully induced within 6 h after refeeding (Fig. 2b). p53 was revealed to be induced mainly in adipocytes when they were isolated from stromal cells by collagenase digestion (Fig. 2c), and the residual stromal cells showed far lower levels of p53 expression which did not rise upon refeeding (data not shown).
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p53 Activation in ob/ob Adipocytes Is Associated with
Induction of p53-regulated GenesTo clarify the potential role of
p53 in ob/ob adipocytes, we further examined the expression
profile of p53 downstream genes such as p21, mdm-2, Bax , and
insulin-like growth factor binding protein-3 (IGFBP-3). As shown in
Fig. 2a, these
p53-regulated genes were elevated in adipose tissue of refed
ob/ob mice. These data indicate that p53 activation causes
the up-regulation of its target genes in ob/ob
adipocytes.
Effects of p53 Absence on the mRNA Expression of p53-regulated Genes in
ob/ob Mouse Adipose TissueTo assess the effects of p53
deficiency in ob/ob mice and validate that the elevation of
p53 downstream genes are really caused by p53, we intercrossed
ob/ob and p53-null mice, and obtained 6 male mice deficient
in both leptin and p53
(ob/obxp53/)
in the C57BL/6J background. Doubly mutant
ob/obxp53/
mice showed no significant difference in body weight, epididymal fat pad
weight, plasma glucose or insulin concentration compared with
ob/ob mice (data not shown). The Northern blot analysis on
these mice in a refed state (Fig.
3) exhibited that p53-regulated genes such as p21, mdm-2, Bax
, and IGFBP-3 in ob/ob mice lacking p53 were
completely suppressed to the same levels as in wild type. These results
established that p53 induction after refeeding caused the elevated expression
of its target genes in ob/ob adipocytes. Heterozygotes of
p53 gene disruption in ob/ob mice maintained the similar
levels of p53 downstream genes to those of ob/ob mice
conceivably by compensation by the intact allele.
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Up-regulation of p21 and IGFBP-3 by Fasting Is Independent of p53In our studies of fasted and refed mice, we also found that p21 and IGFBP-3 are elevated when wild-type mice are fasted. To determine whether these changes are ascribable to p53, we analyzed p53/ mice in fasted or refed conditions. As shown in Fig. 4, the elevation of p21 and IGFBP-3 in a fasted state was also observed in p53/ mice, and hence, entirely independent of p53.
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Defective Refeeding Responses of Lipogenic Genes in ob/ob Mice Is Mediated by p53Lipogenic enzymes such as fatty acid synthase and ATP citrate lyase are known to be markedly induced in adipose tissue and liver when animals are refed after starvation. In contrast, we have previously reported that the adipose tissue of ob/ob mice shows lower levels and defective refeeding responses in the expression of lipogenic enzymes as well as SREBP-1 that regulates their transcription (19). In the current studies we found that the suppression of lipogenic enzymes in ob/ob adipose tissue is confined to a subgroup of enzymes such as fatty acid synthase and ATP citrate lyase, whose expression is primarily dominated by SREBP-1 (Fig. 5a). In contrast, glycerol-3-phosphate dehydrogenase, a key enzyme for glycerogenesis and also important for lipogenesis, was not suppressed in refed ob/ob mouse adipose tissue. Based on the lack of change in its gene expression by the SREBP-1 overexpression, we assume that glycerol-3-phosphate dehydrogenase is not an SREBP-1 target gene.2 These results suggest that p53 suppresses lipogenic gene expression in ob/ob adipocytes by the inhibition of SREBP-1 expression.
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To test this hypothesis, we evaluated the mRNA expression of lipogenic genes in doubly mutant ob/obxp53/ mice. Northern blot analysis displayed that lipogenic enzymes such as fatty acid synthase and ATP citrate lyase along with SREBP-1 were moderately elevated in ob/ob mice lacking p53, showing that the absence of p53 partially de-suppresses lipogenic gene down-regulation (Fig. 5b). These findings demonstrate that p53 is involved in the suppression of lipogenic genes in ob/ob adipocytes. However, the restoration was limited within relatively minor range, suggesting that other factors than p53 are involved in this negative regulation.
Mechanism by Which p53 Suppresses Lipogenic Gene ExpressionTo explore the mechanism by which p53 suppresses lipogenic gene expression, we performed luciferase reporter assays in cultured cells. We used p53-null cell line SaOS-2 as the transfectant. As shown in Fig. 5c, p53 overexpression suppressed the promoter activity of fatty acid synthase gene as well as that of SREBP-1c gene. In contrast, the promoter activity of glycerol-3-phosphate dehydrogenase gene was not suppressed by p53, which corresponds to the in vivo results described above. These findings provide further evidence that p53 suppress lipogenic genes by reducing SREBP-1 expression.
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DISCUSSION |
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Recently it was reported that the phosphatidylinositol 3-kinase-Akt pathway plays an important role in the regulation of p53 via mdm-2 by its enhancement of mdm-2-mediated ubiquitination and degradation of p53 (39). Hence, it is possible that insulin could suppress p53 activation by this pathway. Given that the insulin signaling via phosphatidylinositol 3-kinase and Akt pathway is attenuated in ob/ob adipocytes (40, 41) (i.e. insulin resistance), this mechanism might be involved in the elevation of p53.
Conversely, it is also possible that the p53 activation could cause insulin resistance and that p53 could be positioned as a modifier of insulin signaling. In addition to our present finding that p53 down-regulates SREBP-1 and thereby lipogenesis, p53 is reported to up-regulate the phosphatase PTEN (42), which opposes phosphatidylinositol 3-kinase and has been implicated to be related to insulin resistance (43, 44). These data suggest that p53 could negatively regulate insulin action in a broad spectrum. The concept that p53 could compete with insulin signaling in nutritional regulation is highly probable considering that insulin ancestrally belongs to a growth factor family closely related to proto-oncogenes, whereas p53 is a tumor suppressor gene.
Obesity is defined as an enlargement of adipose tissue mass and, in case of ob/ob mice, is due to both hyperplasia and hypertrophy of adipocytes (4, 5). Therefore, the hyperplastic adipose tissue in obesity could be regarded as a kind of benign tumor. In fact, it has been recently reported that anti-angiogenic agents developed to treat tumors are also effective for obesity (45). It is well known that in many tumors, although mutated, p53 mRNA are expressed at elevated levels (46). The elevation of p53 both in tumors and in ob/ob adipose tissue might result from a common abnormality characteristic of hyperplastic tissue, for instance, low vascularity.
With respect to the consequences of p53 activation in ob/ob adipocytes, we are speculating that, besides insulin signaling, p53 in ob/ob adipocytes might influence cellular turnover processes. Adipocytes in ob/ob mice are reported to be prone to apoptosis (47). Moreover, some markers of immature adipocytes such as adipocyte differentiation-related protein are increased (48). These facts might perhaps imply that the turnover rate of adipocytes is higher and immature cells are relatively increased in obese animals. Thus, although the exact life cycle of adipocytes is not known and precise measurement of their life span needs to be analyzed, p53 might shorten the life span of adipocytes. In keeping with this, it has been recently reported that a transgenic mouse with chronic p53 activation exhibits accelerated aging of organs including adipose tissue (49).
In conclusion, we discovered that p53 is induced upon refeeding in ob/ob adipocytes, a mechanism by which lipogenic genes are negatively regulated. This finding might open up a new aspect of this diverse regulator relating cell growth and nutritional regulation.
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FOOTNOTES |
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¶ 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{at}umin.ac.jp.
1 The abbreviations used are: TNF, tumor necrosis factor; SREBP, sterol
regulatory element-binding protein-1; IGFBP-3, insulin-like growth
factor-binding protein-3; GPDH, glycerol-3-phosphate dehydrogenase.
2 N. Yahagi, unpublished data.
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ACKNOWLEDGMENTS |
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REFERENCES |
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