p53 Activation in Adipocytes of Obese Mice
The tumor suppressor p53 is a transcription factor that activates or represses its target genes after various genotoxic stresses. We have previously shown that sterol regulatory element-binding protein-1 (SREBP-1), a key transcriptional regulator of triglyceride synthesis, and the lipogenic enzymes under its control are markedly suppressed in adipocytes from genetically obese ob/ob mice. Here we demonstrate that p53 and its target genes are highly induced in adipocytes of ob/ob mice in a fed state, leading to the negative regulation of SREBP-1 and thereby lipogenic genes. In fact, disruption of p53 in ob/ob mice completely suppressed the p53-regulated genes to wild-type levels and partially restored expression of lipogenic enzymes. Consistently, reporter gene analysis showed that p53 overexpression suppressed the promoter activity of the SREBP-1c gene and its downstream genes. Thus, the activation of p53 might constitute a negative feedback loop against excess fat accumulation in adipocytes. In conclusion, we discovered a novel role of p53 in the pathophysiology of obesity. The tumor suppressor p53 is a transcription factor that activates or represses its target genes after various genotoxic stresses. We have previously shown that sterol regulatory element-binding protein-1 (SREBP-1), a key transcriptional regulator of triglyceride synthesis, and the lipogenic enzymes under its control are markedly suppressed in adipocytes from genetically obese ob/ob mice. Here we demonstrate that p53 and its target genes are highly induced in adipocytes of ob/ob mice in a fed state, leading to the negative regulation of SREBP-1 and thereby lipogenic genes. In fact, disruption of p53 in ob/ob mice completely suppressed the p53-regulated genes to wild-type levels and partially restored expression of lipogenic enzymes. Consistently, reporter gene analysis showed that p53 overexpression suppressed the promoter activity of the SREBP-1c gene and its downstream genes. Thus, the activation of p53 might constitute a negative feedback loop against excess fat accumulation in adipocytes. In conclusion, we discovered a novel role of p53 in the pathophysiology of obesity. Obesity is a major health problem in industrialized societies, affecting ∼20 – 40% of adults (1Kuczmarski R.J. Flegal K.M. Campbell S.M. Johnson C.L. J. Am. Med. Assoc. 1994; 272: 205-211Crossref PubMed Scopus (2426) Google Scholar). The genetically obese ob/ob mice develop obesity, insulin resistance, and glucose intolerance owing to an inherited deficiency of the appetite-suppressing hormone, leptin (2Ingalls A.M. Dickie M.M. Snell G.D. J. Hered. 1950; 41: 317-318Crossref PubMed Scopus (670) Google Scholar, 3Mayer J. Bates M.W. Dickie M.M. Science. 1951; 113: 746-747Crossref PubMed Scopus (57) Google Scholar, 4Herberg L. Coleman D.L. 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Although the underlying mechanisms that link obesity and defective insulin signaling are as yet undefined, hypertrophied adipocyte-derived cytokines such as tumor necrosis factor (TNF) 1The 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.-α have been reported to be mediators of insulin resistance in obesity (8Hotamisligil G.S. Shargill N.S. Spiegelman B.M. Science. 1993; 259: 87-91Crossref PubMed Scopus (6333) Google Scholar, 9Uysal K.T. Wiesbrock S.M. Marino M.W. Hotamisligil G.S. Nature. 1997; 389: 610-614Crossref PubMed Scopus (1947) Google Scholar). 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 (10Foretz M. Guichard C. Ferre P. Foufelle F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12737-12742Crossref PubMed Scopus (606) Google Scholar). 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. 11Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (3090) Google Scholar, 12Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11041-11048Crossref PubMed Scopus (1122) Google Scholar, 13Brown M.S. Ye J. Rawson R.B. Goldstein J.L. Cell. 2000; 100: 391-398Abstract Full Text Full Text PDF PubMed Scopus (1158) Google Scholar). 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. 14Shimano H. Trends Cardiovasc. Med. 2000; 10: 275-278Crossref PubMed Scopus (70) Google Scholar, 15Shimano H. Prog. Lipid Res. 2001; 40: 439-452Crossref PubMed Scopus (600) Google Scholar, 16Shimano H. Vitam. Horm. 2002; 65: 167-194Crossref PubMed Google Scholar, 17Horton J.D. Goldstein J.L. Brown M.S. J. Clin. Invest. 2002; 109: 1125-1131Crossref PubMed Scopus (3957) Google Scholar). In fact, SREBP-1 and its downstream lipogenic enzymes are drastically induced when fasted animals are refed (18Horton J.D. Bashmakov Y. Shimomura I. Shimano H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5987-5992Crossref PubMed Scopus (545) Google Scholar). These lipogenic genes belong to the group of genes that are induced most strongly by glucose/insulin 2N. Yahagi, unpublished data. 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 (19Yahagi N. Shimano H. Hasty A.H. Matsuzaka T. Ide T. Yoshikawa T. Amemiya-Kudo M. Tomita S. Okazaki H. Tamura Y. Iizuka Y. Ohashi K. Osuga J. Harada K. Gotoda T. Nagai R. Ishibashi S. Yamada N. J. Biol. Chem. 2002; 277: 19353-19357Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar). 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 (20Vogelstein B. Lane D. Levine A.J. Nature. 2000; 408: 307-310Crossref PubMed Scopus (5905) Google Scholar). 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, γ-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 (21Seto E. Usheva A. Zambetti G.P. Momand J. Horikoshi N. Weinmann R. Levine A.J. Shenk T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 12028-12032Crossref PubMed Scopus (465) Google Scholar, 22Mack D.H. Vartikar J. Pipas J.M. Laimins L.A. Nature. 1993; 363: 281-283Crossref PubMed Scopus (309) Google Scholar). The stresses that activate p53 are diverse, ranging from DNA damage to oxidative stress, hypoxia, and heat shock (23Oren M. J. Biol. Chem. 1999; 274: 36031-36034Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar). The cytostatic and cytotoxic effects of TNFα were also demonstrated to be mediated, at least in part, by p53 activation (24Jeoung D.-i. Tang B. Sonenberg M. J. Biol. Chem. 1995; 270: 18367-18373Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 25Donato N.J. Perez M. J. Biol. Chem. 1998; 273: 5067-5072Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 26Rokhlin O.W. Gudkov A.V. Kwek S. Glover R.A. Gewies A.S. Cohen M.B. Oncogene. 2000; 19: 1959-1968Crossref PubMed Scopus (61) Google Scholar). 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 TNFα 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 α, both of which are well-known p53 target genes, are increased from 2- to 3-fold in ob/ob mice (27Soukas A. Cohen P. Socci N.D. Friedman J.M. Genes Dev. 2000; 14: 963-980PubMed Google Scholar). 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. Animals—p53+/–-C57BL/6J (28Livingstone L.R. White A. Sprouse J. Livanos E. Jacks T. Tlsty T.D. Cell. 1992; 70: 923-935Abstract Full Text PDF PubMed Scopus (1318) Google Scholar) and Lep +/ob-C57BL/6J mice were purchased from the Jackson Laboratories (Bar Harbor, Maine). These mice were intercrossed to produce double homozygotes. Genotypes at the p53 loci were determined using the PCR method with primer pairs specific for the wild-type allele of p53 (primers directed against p53 exon 6 and 7; 5′-GGAAATTTGTATCCCGAGTATCTG-3′ and 5′-GTCTTCCAGTGTGATGATGGTAA-3′, respectively) and the mutant allele of p53 (primers directed against neo; 5′-ATGATTGAACAAGATGGATTGC-3′ and 5′-TCGGTCTTGACAAAAAGAACC-3′). Genotypes at the leptin loci were determined by a PCR-based restricted fragment length polymorphism analysis as previously described (19Yahagi N. Shimano H. Hasty A.H. Matsuzaka T. Ide T. Yoshikawa T. Amemiya-Kudo M. Tomita S. Okazaki H. Tamura Y. Iizuka Y. Ohashi K. Osuga J. Harada K. Gotoda T. Nagai R. Ishibashi S. Yamada N. J. Biol. Chem. 2002; 277: 19353-19357Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar). 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. For fasting and refeeding studies, mice were fasted for 24 h or refed for 12 h after 24-h starvation. For the ob/ob×p53 –/– experiment, mice were refed for 12 h following a 24-h fast prior to sacrifice. All animals were sacrificed in an early phase of the light cycle. Nuclear Protein Extraction and Immunoblotting—Nuclear extract protein from white adipose tissue was prepared as described previously (27Soukas A. Cohen P. Socci N.D. Friedman J.M. Genes Dev. 2000; 14: 963-980PubMed Google Scholar). Briefly, fresh adipose tissue (∼3 g pooled from 3–10 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 × g for 10 min, washed once with 25 ml NDS buffer (1% Nonidet P-40), filtered through 70 μm mesh, pelleted at 500 × g for 10 min, resuspended in 1 volume of 1% citric acid, lysed by the addition of 2.5 of 0.1 Tris, and 0.1 and to °C for of protein were to p53 was using a of The at of p53 was using a of of a of was also were using a and using from fat was using and 10 pooled from group were on a 1% and to a For experiments with from adipocytes by was prepared as previously described M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). In fresh adipose tissue was in ml buffer (10 mm at pH mm NaCl, mm KCl, mm mm mm 25 mm 2 mm at °C and with volume of buffer 4 for with at adipocytes were filtered through μm mesh, washed with ml of buffer A, to and with 4 of (10 from mouse was on The for fatty acid 14 and were as described previously H. J.D. Shimomura I. Brown M.S. Goldstein J.L. J. Clin. Invest. PubMed Scopus Google Scholar, H. N. Amemiya-Kudo M. Hasty A.H. Osuga J. Tamura Y. F. Iizuka Y. Ohashi K. Harada K. Gotoda T. Ishibashi S. Yamada N. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). The for p53, Bax α, insulin-like growth factor-binding glycerol-3-phosphate and tumor necrosis factor were prepared by from mouse or adipose tissue The used for PCR were as for p53, primer was 5′-GGAAATTTGTATCCCGAGTATCTG-3′ and primer was for primer was and primer was for primer was and primer was for primer was and primer was for Bax α, primer was and primer was for IGFBP-3, primer was and primer was for GPDH, primer was and primer was for primer was and primer was The were with using DNA The were with the in at °C with the of p53 and for which buffer was used at The were washed in at were to gene of the mouse SREBP-1c promoter and of the fatty acid synthase promoter were prepared as described previously M. Shimano H. Yoshikawa T. N. Hasty A.H. Okazaki H. Tamura Y. F. Iizuka Y. Ohashi K. Osuga J. Harada K. Gotoda T. R. S. Ishibashi S. Yamada N. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, H. J.D. Shimomura I. Brown M.S. Goldstein J.L. J. Clin. Invest. 1997; PubMed Scopus Google Scholar). was by PCR of to of the mouse glycerol-3-phosphate promoter M. P. H. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and of the PCR a reporter 14 of and the gene was purchased from p53 expression by the promoter was by a DNA fragment with PCR using first from mouse adipose tissue as a and and DNA S. D.L. H. H. J. Biol. Chem. Full Text PDF PubMed Google Scholar). All DNA with PCR were by and human cell was from the cells were in 25 mm and μg/ml supplemented with under at cells were on a 12 well at × reporter and a 0.1 were cells using 6 to the a the of was of cells were and the of activity in was by standard p53 upon in ob/ob at the our first of we demonstrated that ob/ob mouse adipose tissue levels of p53 and protein p53 is known to be at levels including through protein and and we found that the of p53 activation in ob/ob mouse adipose tissue was at this not with the induced an of p53, at was also in ob/ob adipose tissue we the of p53 under various our the increased expression of p53 in ob/ob mouse adipose tissue was to a fed and was in a fasted The of p53 to be induced 6 h after refeeding p53 was revealed to be induced in adipocytes when were from cells by and the cells showed levels of p53 expression which not upon refeeding not p53 in ob/ob with of p53-regulated the role of p53 in ob/ob we examined the expression profile of p53 downstream genes such as Bax α, and insulin-like growth factor binding shown in these p53-regulated genes were in adipose tissue of refed ob/ob mice. These that p53 activation causes the of its target genes in ob/ob adipocytes. of p53 on the of p53-regulated Genes in ob/ob the effects of p53 deficiency in ob/ob mice and that the of p53 downstream genes are by p53, we intercrossed ob/ob and mice, and 6 male mice in both leptin and p53 in the mutant ob/ob×p53 –/– mice showed in fat glucose or insulin concentration with ob/ob mice not The analysis on these mice in a refed that p53-regulated genes such as Bax α, and in ob/ob mice lacking p53 were completely suppressed to the levels as in These established that p53 after refeeding the expression of its target genes in ob/ob adipocytes. of p53 gene disruption in ob/ob mice the levels of p53 downstream genes to of ob/ob mice by by the of p21 and by of our of fasted and refed mice, we also found that p21 and are when wild-type mice are these are to p53, we mice in fasted or refed shown in the of p21 and in a fasted was also in mice, and of of Genes in ob/ob Mice by enzymes such as fatty acid synthase and are known to be markedly induced in adipose tissue and when animals are refed after starvation. In we have previously reported that the adipose tissue of ob/ob mice levels and defective refeeding responses in the expression of lipogenic enzymes as well as SREBP-1 that transcription (19Yahagi N. Shimano H. Hasty A.H. Matsuzaka T. Ide T. Yoshikawa T. Amemiya-Kudo M. Tomita S. Okazaki H. Tamura Y. Iizuka Y. Ohashi K. Osuga J. Harada K. Gotoda T. Nagai R. Ishibashi S. Yamada N. J. Biol. Chem. 2002; 277: 19353-19357Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar). In the we found that the suppression of lipogenic enzymes in ob/ob adipose tissue is to a of enzymes such as fatty acid synthase and expression is by SREBP-1 In glycerol-3-phosphate a key for and also for was not suppressed in refed ob/ob mouse adipose Based on the of in its gene expression by the SREBP-1 we that glycerol-3-phosphate is not an SREBP-1 target These that p53 suppresses lipogenic gene expression in ob/ob adipocytes by the of SREBP-1 this we the expression of lipogenic genes in mutant ob/ob×p53 –/– mice. analysis that lipogenic enzymes such as fatty acid synthase and with SREBP-1 were in ob/ob mice lacking p53, that the absence of p53 partially lipogenic gene down-regulation These demonstrate that p53 is involved in the suppression of lipogenic genes in ob/ob adipocytes. However, the was that other factors p53 are involved in this negative regulation. by p53 the mechanism by which p53 suppresses lipogenic gene we performed reporter in We used cell as the shown in p53 overexpression suppressed the promoter activity of fatty acid synthase gene as well as that of SREBP-1c In the promoter activity of glycerol-3-phosphate gene was not suppressed by p53, which to the in described These that p53 lipogenic genes by SREBP-1 present demonstrate that p53 is induced upon refeeding in ob/ob leading to the negative regulation of lipogenic genes. The mechanisms of p53 are currently not However, the causes that to p53 activation are diverse, ranging from DNA damage to oxidative and (23Oren M. J. Biol. Chem. 1999; 274: 36031-36034Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar). We that ob/ob adipocytes various stresses that could induce p53 The of the expression of which is increased in ob/ob adipocytes (8Hotamisligil G.S. Shargill N.S. Spiegelman B.M. Science. 1993; 259: 87-91Crossref PubMed Scopus (6333) Google Scholar), could to The of cell volume might that could activate in a fed ob/ob mice are markedly which also to in oxidative through various mechanisms K. M. Y. T. M. C. 1993; PubMed Scopus Google Scholar, T. P. F. M. M. T. T. T. M. H. H. H. 2000; PubMed Scopus Google Scholar, H. K. S. A. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, ob/ob adipocytes are presumably under p53 can be activated. it was reported that the plays an role in the regulation of p53 by its of and of p53 Y. S. T. Y. T. K. N. Y. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). it is possible that insulin could p53 activation by this that the insulin signaling and is in ob/ob adipocytes J. H. L. Friedman J.E. J. 2000; PubMed Scopus Google Scholar, E. C. U. 2000; PubMed Google Scholar) insulin this mechanism might be involved in the of it is also possible that the p53 activation could insulin resistance and that p53 could be as a of insulin signaling. In addition to our present that p53 SREBP-1 and thereby p53 is reported to the D. D. Y. B. Y. S. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), which and has been to be related to insulin resistance N. T. R. J.M. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, M. R.A. D. S. 2002; PubMed Scopus Google Scholar). These that p53 could regulate insulin in a The that p53 could with insulin signaling in regulation is highly that insulin to a growth factor family related to p53 is a tumor suppressor Obesity is as an of adipose tissue in of ob/ob mice, is to both and of adipocytes L. Coleman D.L. Metabolism. 1977; 26: 59-99Abstract Full Text PDF PubMed Scopus (362) Google Scholar, 5Bray G.A. York D.A. Physiol. Rev. 1979; 59: 719-809Crossref PubMed Scopus (986) Google Scholar). the adipose tissue in obesity could be regarded as a of In fact, it has been recently reported that to are also for obesity M.A. D. S.M. R. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar). It is well known that in p53 are at levels E. D. Res. Google Scholar). The of p53 both in and in ob/ob adipose tissue might from a of for to the of p53 activation in ob/ob we are insulin p53 in ob/ob adipocytes might cellular in ob/ob mice are reported to be to Lane Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar). Moreover, of adipocytes such as protein are increased J.P. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). These might that the of adipocytes is and cells are increased in obese animals. Thus, the cycle of adipocytes is not known and precise of to be p53 might the of adipocytes. In with it has been recently reported that a mouse with p53 activation of including adipose tissue S. J. S. N. H. B. C. S. T. A. L.A. Nature. 2002; PubMed Scopus Google Scholar). In conclusion, we discovered that p53 is induced upon refeeding in ob/ob a mechanism by which lipogenic genes are might a of this regulator cell growth and regulation. We H. Hasty for of the