Cyclin-dependent Kinase Inhibitor, p21WAF1/CIP1, Is Involved in Adipocyte Differentiation and Hypertrophy, Linking to Obesity, and Insulin Resistance

Abstract

Both adipocyte hyperplasia and hypertrophy are determinant factors for adipocyte differentiation during the development of obesity. 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 and in 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 p21-/- mice impaired adipocyte differentiation, resulting in smaller adipocytes. Despite normal adipose tissue mass on a normal diet, p21-/- 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 by activating 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 to increased downstream pathophysiological consequences such as insulin resistance. Both adipocyte hyperplasia and hypertrophy are determinant factors for adipocyte differentiation during the development of obesity. 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 and in 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 p21-/- mice impaired adipocyte differentiation, resulting in smaller adipocytes. Despite normal adipose tissue mass on a normal diet, p21-/- 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 by activating 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 to increased downstream pathophysiological consequences such as insulin resistance. The process of adipocyte differentiation has been extensively characterized in cultures of preadipocyte clonal cell lines such as mouse 3T3-L1 and 3T3-F442A (1Gregoire F.M. Smas C.M. Sul H.S. Physiol. Rev. 1998; 78: 783-809Crossref PubMed Scopus (1834) Google Scholar, 2Rosen E.D. Spiegelman B.M. Annu. Rev. Cell Dev. Biol. 2000; 16: 145-171Crossref PubMed Scopus (1046) Google Scholar). Adipogenesis requires a 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 is reasonable to assume that factors involved in cell cycle regulation may have important roles in the adipocyte differentiation process. Cell cycle progression in mammals is governed by various complexes of cyclins and cyclin-dependent kinases (CDKs) 2The abbreviations used are: CDK, cyclin-dependent kinase; RNAi, RNA-mediated interference; MEF, mouse embryonic fibroblast; HFHS, high fat high sucrose; PPAR, peroxisome proliferator-activated receptor; DEXA, dual energy x-ray absorptiometry; TUNEL, terminal dUTP nick-end labeling; C/EBP, CCAAT/enhancer-binding protein. 2The abbreviations used are: CDK, cyclin-dependent kinase; RNAi, RNA-mediated interference; MEF, mouse embryonic fibroblast; HFHS, high fat high sucrose; PPAR, peroxisome proliferator-activated receptor; DEXA, dual energy x-ray absorptiometry; TUNEL, terminal dUTP nick-end labeling; C/EBP, CCAAT/enhancer-binding protein. as well as their inhibitors, of which p21 and p27 are the most widely studied. It was previously suggested that expression of p21 and p27 is changed during the adipogenesis (3Morrison R.F. Farmer S.R. J. Biol. Chem. 1999; 274: 17088-17097Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). 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 (3Morrison R.F. Farmer S.R. J. Biol. Chem. 1999; 274: 17088-17097Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar, 4Nakae J. Kitamura T. Kitamura Y. Biggs W.H. II I Arden K.C. Accili D. Dev. Cell. 2003; 4: 119-129Abstract Full Text Full Text PDF PubMed Scopus (596) Google Scholar, 5Sakai T. Sakaue H. Nakamura T. Okada M. Matsuki Y. Watanabe E. Hiramatsu R. Nakayama K. Nakayama K.I. Kasuga M. J. Biol. Chem. 2007; 282: 2038-2046Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). 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 physical activity 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 cardiovascular diseases (6Spiegelman B.M. Flier J.S. Cell. 2001; 104: 531-543Abstract Full Text Full Text PDF PubMed Scopus (1919) Google Scholar). Impaired adipocyte differentiation is also related to insulin resistance. Based upon the potential roles of CDK inhibitors in cell cycle progression and apoptosis, it is reasonable to speculate that CDK inhibitors could be involved in obesity and insulin 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. 7Harper J.W. Adami G.R. Wei N. Keyomarsi K. Elledge S.J. Cell. 1993; 75: 805-816Abstract Full Text PDF PubMed Scopus (5222) Google Scholar, 8el-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7903) Google Scholar, 9Xiong Y. Hannon G.J. Zhang H. Casso D. Kobayashi R. Beach D. Nature. 1993; 366: 701-704Crossref PubMed Scopus (3163) Google Scholar, 10Noda A. Ning Y. Venable S.F. Pereira-Smith O.M. Smith J.R. Exp. Cell Res. 1994; 211: 90-98Crossref PubMed Scopus (1313) Google Scholar; for review, see Refs. 11Hunter T. Cell. 1993; 75: 839-841Abstract Full Text PDF PubMed Scopus (370) Google Scholar and 12Polyak K. Waldman T. He T.C. Kinzler K.W. Vogelstein B. Genes Dev. 1996; 10: 1945-1952Crossref PubMed Scopus (473) Google Scholar). p21 is a well known target of p53, a stress response. In addition to causing G1 arrest, p21 protects cells from apoptosis (12Polyak K. Waldman T. He T.C. Kinzler K.W. Vogelstein B. Genes Dev. 1996; 10: 1945-1952Crossref PubMed Scopus (473) Google Scholar). We recently found that p21 is a direct gene target of SREBPs, crucial transcription factors involved in lipogenesis and adipogenesis (13Inoue N. Shimano H. Nakakuki M. Matsuzaka T. Nakagawa Y. Yamamoto T. Sato R. Takahashi A. Sone H. Yahagi N. Suzuki H. Toyoshima H. Yamada N. Mol. Cell. Biol. 2005; 25: 8938-8947Crossref PubMed Scopus (52) Google Scholar, 14Shimomura I. Hammer R.E. Richardson J.A. Ikemoto S. Bashmakov Y. Goldstein J.L. Brown M.S. Genes Dev. 1998; 12: 3182-3194Crossref PubMed Scopus (673) Google Scholar). We also reported that p21 expression was highly up-regulated in hypertrophic adipose tissue and in the liver in models of obesity (15Yahagi N. Shimano H. Matsuzaka T. Najima Y. Sekiya M. Nakagawa Y. Ide T. Tomita S. Okazaki H. Tamura Y. Iizuka Y. Ohashi K. Gotoda T. Nagai R. Kimura S. Ishibashi S. Osuga J. Yamada N. J. Biol. Chem. 2003; 278: 25395-25400Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 16Yahagi N. Shimano H. Matsuzaka T. Sekiya M. Najima Y. Okazaki S. Okazaki H. Tamura Y. Iizuka Y. Inoue N. Nakagawa Y. Takeuchi Y. Ohashi K. Harada K. Gotoda T. Nagai R. Kadowaki T. Ishibashi S. Osuga J. Yamada N. J. Biol. Chem. 2004; 279: 20571-20575Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). These data prompted us to explore the role of p21 in adipocyte differentiation and hypertrophy and to determine the involvement of p21 in obesity and insulin resistance. 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-coated dishes (BD Biosciences) (day 4) and cultured to confluence (day 2). After 2 days, cells were replaced with differentiation induction medium A (5 μg/ml insulin, 1 μm dexamethasone, and 0.5 mm isobutylmethylxanthine) (day 0). After 48 h cells were changed 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) were constructed with BLOCK-iT™ U6 RNAi Entry Vector kit and BLOCK-iT™ Adenoviral RNAi Expression system (Invitrogen). Each adenoviral vector was propagated in 293A cells and purified by cesium chloride density centrifugation (17Ide T. Shimano H. Yahagi N. Matsuzaka T. Nakakuki M. Yamamoto T. Nakagawa Y. Takahashi A. Suzuki H. Sone H. Toyoshima H. Fukamizu A. Yamada N. Nat. Cell Biol. 2004; 6: 351-357Crossref PubMed Scopus (278) Google Scholar, 18Nakagawa Y. Shimano H. Yoshikawa T. Ide T. Tamura M. Furusawa M. Yamamoto T. Inoue N. Matsuzaka T. Takahashi A. Hasty A.H. Suzuki H. Sone H. Toyoshima H. Yahagi N. Yamada N. Nat. Med. 2006; 12: 107-113Crossref PubMed Scopus (92) Google Scholar). 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 (19Okazaki H. Osuga J. Tamura Y. Yahagi N. Tomita S. Shionoiri F. Iizuka Y. Ohashi K. Harada K. Kimura S. Gotoda T. Shimano H. Yamada N. Ishibashi S. Diabetes. 2002; 51: 3368-3375Crossref PubMed Scopus (99) Google Scholar). 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 and The by J. Beach D. T. Hannon G.J. Nature. PubMed Scopus Google and were the for or were by to by The were maintained on a normal diet for a diet-induced the mice were fed on a high fat high diet as previously N. I. K. H. M. H. N. H. Takahashi M. Y. R. N. S. Y. K. M. S. T. T. Y. Nat. Med. 2002; PubMed Scopus Google at 10 of for We performed of for of insulin, and and were to glucose the diet insulin the diet dual energy x-ray and at were fixed in 10% formalin for and in for the was used to tissue and fat mass A. A. H. J. 2004; PubMed Scopus Google Scholar). of Adipocyte and fat from normal mice and mice were fixed in for of from to μm in normal mice or from to μm in mice were by through and and of adipocytes were with a for normal mice and a for mice as previously A. A. H. J. 2004; PubMed Scopus Google Scholar, J. E. J. Res. Full Text PDF PubMed Google Scholar, J. Res. Full Text PDF PubMed Google Scholar). were performed on mice were at 2 of glucose and insulin were and 60 min were performed on mice for were with insulin at 0.5 or of and glucose was min with II and were with and from insulin and were with the mouse insulin kit and the mouse kit of was from adipose tissue, adipose tissue, and cultured cells (Invitrogen). of from mice and cells were of with and to in a and to Biosciences) for were with The were with the in Biosciences) at °C and washed in × at were to and with Cell of cell a and kit I was used to the the of apoptosis, the In Cell kit was The of this kit was by terminal which the of to in a were in mm mm mm mm 1 mm and inhibitor were the kit were on or by to were in at °C with of p53, of p53, of and After with were by or kit In into of feeding, mice were by before tissue The adenovirus × was on of the epididymal fat T. H. Y. T. J. K. Y. J. H. A. H. H. T. Y. Cell 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). are as the was the are by of the of p21 on Adipocyte in fibroblasts were differentiated into and p21 expression was with was a induction of p21 at the time of differentiation into adipocytes (3Morrison R.F. Farmer S.R. J. Biol. Chem. 1999; 274: 17088-17097Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). Expression of p21 was at both the and during terminal differentiation and during the hypertrophic phase of lipid determine the of p21 knockdown of p21 adenoviral with RNAi and before induction of differentiation was performed p21 caused a of adipocyte differentiation as by Oil Red O The of p21 in in p27 in p21 was accompanied by reduced of and transcription factors involved in adipocyte differentiation, with a for adipogenesis known to at of adipogenesis was by p21 that the absence of p21 the of adipocyte differentiation of the of p21 during Adipocyte in determine the of absence of mouse embryonic fibroblasts from p21-/- mice were induced to into adipocytes differentiation, p21-/- cells increased cell and of as with cell growth in the absence of p21 A and induction of differentiation, p21-/- cells to however, at terminal differentiation the size of the adipocytes was smaller differentiated type These data that p21 is for adipocyte of the of p21 in with the from cultured of p21-/- mice was were p21-/- and mice fat and adipocyte size changed with a a in p21-/- mice B and and were in insulin, or and in p21-/- mice as by glucose and insulin and and the role of p21 in adipogenesis in diet-induced obesity was data that gene expression of p21 was highly induced by high fat diet, a role of this in the hypertrophic of adipocytes p21-/- mice were fed the diet, which obesity and insulin the of was with The in p21-/- mice was to decreased fat mass as by The of epididymal fat was also decreased in p21-/- mice fat of mice was of and p21-/- the of p21 on The size of adipocytes from the epididymal adipose tissue, as by a also a in the size of p21-/- adipocytes of adipose tissue that the marked of adipocytes in mice was in p21-/- mice The of lipid which in the of fat cells of was decreased in p21-/- mice In adipose tissue of p21-/- mice fed diets to mice on a diet and The of obesity was accompanied by marked improvement of insulin resistance insulin in p21-/- mice was by decreased insulin or of glucose in and fed and in glucose A and a in glucose of insulin in p21-/- mice in mice These data that gene of p21 a from diet-induced obesity and a marked improvement in insulin resistance. from diet-induced obesity was to in for and p21-/- data were a Expression in of p21-/- of various in adipose tissue of and p21-/- mice were by transcription factors such as and were and p21-/- Expression of and known to be up-regulated in adipose tissue in was in p21-/- mice on a diet, known to be in was up-regulated in p21-/- adipose to by decreased and increased were with of obesity in p21-/- mice on a diet Expression of 1 and was also that could be by the absence of p21 in adipose tissue, in the and in are known to be involved in energy These were decreased in diet-induced obesity in and their were in p21-/- a of energy to of in by p21 against determine the by which the absence of p21 protects against diet-induced obesity and insulin p21 was in 3T3-L1 cells had differentiated into adipocytes. In to of adenoviral RNAi before the differentiation of p21 at caused of lipid of the and a marked in of the medium that these apoptosis B and these that of p21 was by a induction of the terminal of apoptosis, in expression of a gene that a role from apoptosis at with apoptosis K. H. T. K. T. H. K. Tokino T. Nakamura Y. Y. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google was induced at 48 h with adenovirus These data that p21 during the terminal differentiation of adipocytes could apoptosis, the hypertrophic for of p53, the most of is known to be in to has been in 3T3-L1 cells and remains during accumulation of lipids in with the S.J. D. D. 1999; PubMed Scopus Google Scholar, C.M. Mol. Cell. Biol. 1996; 16: PubMed Google Scholar). of at and is the process for p21 J. K. Y. Genes Dev. 2000; PubMed Google Scholar, 1999; PubMed Scopus Google Scholar). In with p21 the of these was during the adipocyte In of at was reported to be with apoptosis K. H. T. K. T. H. K. Tokino T. Nakamura Y. Y. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). of was induced at the of differentiation in the 3T3-L1 adipocytes the and of by p21 knockdown in 3T3-L1 adipocytes of of and p21 in 3T3-L1 cells during adipocyte 3T3-L1 fibroblasts were differentiated into adipocytes to the The of p53, p21 and of at and crucial for p21 were at the by 3T3-L1 cells were induced to into adipocytes and with p21 RNAi adenovirus at of p53, of p53, and a in 3T3-L1 adipocytes at the the induction for adipocyte differentiation were by the involvement of apoptosis in the of p21 in adenoviral RNAi for p21 was into epididymal adipose tissue of of p21 expression was the causing a to of accompanied by decreased glucose and insulin in insulin and glucose and induction of adipocyte apoptosis by p21 knockdown These data implicated that p21 could be induced by diet-induced obesity and to from apoptosis during adipocyte hypertrophy. of p21 in Adipocyte and that p21 a crucial role in of differentiation and hypertrophy of adipocytes. p21 is induced during adipogenesis and during terminal p21 knockdown that p21 to is for adipocyte of vigorous apoptosis upon p21 knockdown during the terminal phase of 3T3-L1 differentiation and in hypertrophic adipose tissue during diet-induced obesity in mice that p21 is crucial for adipocyte hypertrophy for the of In p21 was highly induced in enlarged adipose tissue of diet-induced obese and its absence obesity. In to knockdown of apoptosis was in adipose tissue from p21-/- mice on a diet or in p21-/- adipocyte differentiation We speculate that in the absence of adipocytes hypertrophic to apoptosis. In normal it is that the increased accumulation of which during differentiation, is a stress to leading to of the In this p21 be for of hypertrophic adipocytes. potential of p21 is through its its growth arrest It has been that adipocyte hypertrophy apoptosis A. H. K. K. Y. K. K. Y. T. H. Y. Kadowaki T. J. 1998; PubMed Scopus Google Scholar, C.M. K. S. Diabetes. PubMed Google of apoptosis was data that in the of hypertrophic adipocytes are against apoptosis, this of in p21 Expression in have that the transcription factors and are involved in adipocyte differentiation, and have activity for the p21 J. Kitamura T. Kitamura Y. Biggs W.H. II I Arden K.C. Accili D. Dev. Cell. 2003; 4: 119-129Abstract Full Text Full Text PDF PubMed Scopus (596) Google Scholar, N. Shimano H. Nakakuki M. Matsuzaka T. 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Cell. 2000; Full Text Full Text PDF PubMed Scopus Google that was by data that hypertrophic adipocytes in obesity are also under stress and are to apoptosis, which under normal could be by of Thus, we that p21 a role in adipogenesis by cell cycle by the apoptosis of hypertrophic adipocytes. of p21 on Obesity and of high fat diet-induced obesity in p21-/- mice was with insulin sensitivity. in p21-/- mice was originally reported to be increased as by epididymal fat A. A. H. J. 2004; PubMed Scopus Google Scholar). The of this with data is however, we speculate that it could be in which we used p21-/- mice to the on mice on the We that p21 in adipocytes the major role in obesity and insulin resistance knockdown of p21 in epididymal fat the apoptosis and decreased the size of adipocytes as in p21-/- mice on decreased size of adipose tissue was with in 1 and expression and in obesity were in p21-/- The absence of p21 in could to of in adipose as reported in 2004; PubMed Scopus Google Scholar). were in the expression of related to energy in liver and of p21-/- mice on diet, that are the for in the absence of It be that in adipose tissue, was in p21-/- obesity is accompanied by fat accumulation and decreased in this tissue (6Spiegelman B.M. Flier J.S. Cell. 2001; 104: 531-543Abstract Full Text Full Text PDF PubMed Scopus (1919) Google which was in p21-/- mice the and p21-/- it is that p21 to of diet-induced obesity and insulin resistance at by the of fat A Cell and that factors for the regulation of cell growth and are also involved in A on a direct p21 and this a CDK inhibitor and D. A. Mol. Cell. Biol. 2006; PubMed Scopus Google Scholar). The a role in the of hypertrophic adipocytes in of excess In the of diseases including insulin resistance and of p21 in adipocytes have a by adipose tissue it is also adipocyte differentiation is important for storage of excess lipids in adipose tissue, protecting against lipid storage and insulin resistance. are to the precise by which is and involved in obesity and insulin resistance in adipose on CDK inhibitor, as well as the that of the in the cell of adipocytes and cells T. Sakaue H. Nakamura T. Okada M. Matsuki Y. Watanabe E. Hiramatsu R. Nakayama K. Nakayama K.I. Kasuga M. J. Biol. 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Diabetes. 2006; PubMed Scopus Google Scholar). these data p21 in adipocyte cell size and p27 in cell and adipose tissue growth by both of the major CDK In data that p21 dual roles in both adipocyte differentiation through cell cycle arrest and in adipocyte hypertrophy through its Via these p21 adipose tissue expansion during obesity to insulin resistance. data that a cell cycle can be involved in a of and in a cell growth and We are to Sakaue and Nakamura for of adipocyte cell size by We for of with