Skp2 Controls Adipocyte Proliferation during the Development of Obesity

The increase in the mass of adipose tissue during the development of obesity can arise through an increase in cell size, an increase in cell number, or both. Here we show that long term maintenance of C57BL/6 mice on a high fat diet (for ∼25 weeks) induces an initial increase in adipocyte size followed by an increase in adipocyte number in white adipose tissue. The latter effect was found to be accompanied by up-regulation of expression of the gene for the F-box protein Skp2 as well as by downregulation of the cyclin-dependent kinase inhibitor p27Kip1, a principal target of the SCFSkp2 ubiquitin ligase, in white adipose tissue. Ablation of Skp2 protected mice from the development of obesity induced either by a high fat diet or by the lethal yellow agouti (Ay) mutation, and this protective action was due to inhibition of the increase in adipocyte number without an effect on adipocyte hypertrophy. The reduction in the number of adipocyte caused by Skp2 ablation also inhibited the development of obesity-related insulin resistance in the Ay mutant mice, although the reduced number of β cells and reduced level of insulin secretion in Skp2-deficient mice resulted in glucose intolerance. Our observations thus indicate that Skp2 controls adipocyte proliferation during the development of obesity. The increase in the mass of adipose tissue during the development of obesity can arise through an increase in cell size, an increase in cell number, or both. Here we show that long term maintenance of C57BL/6 mice on a high fat diet (for ∼25 weeks) induces an initial increase in adipocyte size followed by an increase in adipocyte number in white adipose tissue. The latter effect was found to be accompanied by up-regulation of expression of the gene for the F-box protein Skp2 as well as by downregulation of the cyclin-dependent kinase inhibitor p27Kip1, a principal target of the SCFSkp2 ubiquitin ligase, in white adipose tissue. Ablation of Skp2 protected mice from the development of obesity induced either by a high fat diet or by the lethal yellow agouti (Ay) mutation, and this protective action was due to inhibition of the increase in adipocyte number without an effect on adipocyte hypertrophy. The reduction in the number of adipocyte caused by Skp2 ablation also inhibited the development of obesity-related insulin resistance in the Ay mutant mice, although the reduced number of β cells and reduced level of insulin secretion in Skp2-deficient mice resulted in glucose intolerance. Our observations thus indicate that Skp2 controls adipocyte proliferation during the development of obesity. The worldwide epidemic of obesity is a serious threat to public health, in part because the increase in the mass of white adipose tissue (WAT) 2The abbreviations used are: WAT, white adipose tissue; CDK, cyclin-dependent kinase; CKI, cyclin-dependent kinase inhibitor; Rb, retinoblastoma protein; SCF, Skp1-Cullin-F-box protein; PDK1, phosphoinositide-dependent kinase 1; Erk, extracellular signal-regulated kinase; E3, ubiquitin-protein isopeptide ligase. in obese individuals increases the risk for development of insulin resistance and type 2 diabetes mellitus (1Yach D. Stuckler D. Brownell K.D. Nat. Med. 2006; 12: 62-66Crossref PubMed Scopus (638) Google Scholar). The expansion of WAT during the development of obesity can occur through increases in cell number (adipocyte hyperplasia) and in cell size (adipocyte hypertrophy) (2Haslam D.W. James W.P. Lancet. 2005; 366: 1197-1209Abstract Full Text Full Text PDF PubMed Scopus (3674) Google Scholar, 3Hausman D.B. DiGirolama M. Bartness T.J. Hausman G.J. Martin R.J. Obesity Rev. 2001; 2: 239-254Crossref PubMed Scopus (530) Google Scholar). However, the precise contribution of adipocyte number to the pathogenesis of obesity and obesity-related insulin resistance remains unclear. The number of adipocytes is thought to increase as a result of the proliferation of preadipocytes and their subsequent differentiation into mature adipocytes (4Bjorntorp P. Horm. Metab. Res. 1974; 4: 77-83PubMed Google Scholar, 5Spiegelman B.M. Flier J.S. Cell. 1996; 87: 377-389Abstract Full Text Full Text PDF PubMed Scopus (1173) Google Scholar). Both the proliferation and differentiation of preadipocytes are characterized by marked changes in the pattern of gene expression that are achieved by the sequential induction of various transcription factors, including members of the CCAAT/enhancer-binding protein (C/EBP-α,-β, and -δ), peroxisome proliferator-activated receptor (PPAR-γ,-β, and -δ), basic helix-loop-helix (SREBP-1c), and Kruppel-like zinc-finger factor (KLF-5 and -15) families (6Oishi Y. Manabe I. Tobe K. Tsushima K. Shindo T. Fujiu K. Nishimura G. Maemura K. Yamauchi T. Kubota N. Suzuki R. Kitamura T. Akira S. Kadowaki T. Nagai R. Cell Metab. 2005; 1: 27-39Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar, 7Mori T. Sakaue H. Iguchi H. Gomi H. Okada Y. Takashima Y. Nakamura K. Nakamura T. Yamauchi T. Kubota N. Kadowaki T. Matsuki Y. Ogawa W. Hiramatsu R. Kasuga M. J. Biol. Chem. 2005; 280: 12867-12875Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). These proteins are thought to act synergistically in the transcriptional activation of a variety of adipocytespecific genes, with each also reciprocally activating the expression of the others (8Lane M.D. Tang Q.Q. Jiang M.S. Biochem. Biophys. Res. Commun. 1999; 266: 677-683Crossref PubMed Scopus (243) Google Scholar, 9Wu Z. Rosen E.D. Brun R. Hauser S. Adelmant G. Troy A.E. McKeon C. Darlington G.J. Spiegelman B.M. Mol. Cell. 1999; 3: 151-158Abstract Full Text Full Text PDF PubMed Scopus (862) Google Scholar). In addition to transcription factors, the cell cycle plays an important role in adipogenesis, given that inhibition of DNA synthesis prevents the differentiation of 3T3-L1 preadipocytes into adipocytes (10Patel Y.M. Lane M.D. J. Biol. Chem. 2000; 275: 17653-17660Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Cell proliferation is regulated at each phase of the cell cycle by the activation and deactivation of various cell cycle-related proteins, including cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors (CKIs) (11Morgan D.O. Annu. Rev. Cell Dev. Biol. 1997; 13: 261-291Crossref PubMed Scopus (1833) Google Scholar, 12Sherr C.J. Roberts J.M. Genes Dev. 2002; 13: 1501-1512Crossref Scopus (5220) Google Scholar). CDK4 and CDK2 regulate the transition from G1 to S phase of the cell cycle as well as progression of S phase by phosphorylating substrates such as the retinoblastoma protein (Rb) (13Tsai L.-H. Harlow E. Meyerson M. Nature. 1991; 353: 174-177Crossref PubMed Scopus (415) Google Scholar, 14Elledge S.J. Spottswood R. EMBO J. 1991; 10: 2653-2659Crossref PubMed Scopus (219) Google Scholar). Phosphorylation of Rb induces its dissociation from the E2F1 complex, the latter of which then promotes cell cycle progression (15Lundberg A.S. Weinberg R.A. Mol. Cell. Biol. 1998; 18: 753-761Crossref PubMed Scopus (872) Google Scholar). Evidence suggests that CDK2 (16Tang Q.Q. Otto T.C. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 44-49Crossref PubMed Scopus (658) Google Scholar), CDK4 (17Abella A. Dubus P. Malumbres M. Rane S.G. Kiyokawa H. Sicard A. Vignon F. Langin D. Barbacid M. Fajas L. Cell Metab. 2005; 2: 239-249Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), Rb (18Hansen J.B. Petersen R.K. Jorgensen C. Kristiansen K. J. Biol. Chem. 2002; 277: 26335-26339Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), and E2F1 (19Fajas L. Landsberg R.L. Huss-Garcia Y. Sardet C. Lees J.A. Auwerx J. Dev. Cell. 2002; 3: 39-49Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar) are essential not only for cell proliferation but also for differentiation during adipogenesis. CKIs include two families of proteins, the Cip (Kip) family and Ink4 family, and are central players in the exit of cells from the cell cycle (20Nakayama K.I. Nakayama K. Nature Rev. Cancer. 2006; 6: 369-381Crossref PubMed Scopus (1189) Google Scholar). The loss of p27Kip1 or p21Cip1 in mice leads to adipocyte hyperplasia as a result of increased proliferation or recruitment of preadipocytes (21Naaz A. Holsberger D.R. Iwamoto G.A. Nelson A. Kiyokawa H. Cooke P.S. FASEB J. 2004; 18: 1925-1927Crossref PubMed Scopus (121) Google Scholar), suggesting that these CKIs are important in regulation of adipocyte number. The SCF (Skp1-Cullin-F-box protein) ubiquitin ligase (E3) complex targets CKIs for degradation by the 26 S proteasome and thereby regulates cell cycle progression (22Zhang H. Kobayashi R. Galaktionov K. Beach D. Cell. 1995; 82: 915-925Abstract Full Text PDF PubMed Scopus (451) Google Scholar). Skp2, the substrate-binding subunit of the SCFSkp2 complex, contributes to the degradation of p27Kip1, an inhibitor of CDK2 and CDK1 activities that promote entry into S phase and mitosis, respectively. The abundance of Skp2 is increased in various human cancers, in which its expression is inversely related to that of p27Kip1 (23Pagano M. Benmaamar R. Cancer Cell. 2003; 4: 251-256Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). We have now investigated the role of Skp2 in adipogenesis and show here that the amount of Skp2 mRNA is increased in WAT during the development of obesity in mice. Our genetic analyses further suggest that Skp2 controls adipocyte proliferation during the development of this condition in mice. Antibodies and Other Reagents—Antibodies to Skp2 and to Erk1 or Erk2 (Erk1/2) were obtained from Zymed Laboratories Inc. (San Francisco, CA) and Cell Signaling Technologies (Beverly, MA), respectively. Antibodies to p27Kip1 and to cyclin E1 were from Santa Cruz Biotechnology (Santa Cruz, CA). An adenovirus encoding Skp2 was generated as described previously (24Nakayama K. Nagahama H. Minamishima Y.A. Miyake S. Ishida N. Hatakeyama S. Kitagawa M. Iemura S. Natsume T. Nakayama K.I. Dev. Cell. 2004; 6: 661-672Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). Animals—Male C57BL/6 and KKAy/TaJcl mice were obtained from CLEA Japan. Skp2 knock-out mice were generated as described previously (25Nakayama K. Nagahama H. Minamishima Y.A. Matsumoto M. Nakamichi I. Kitagawa K. Shirane M. Tsunematsu R. Tsukiyama T. Ishida N. Kitagawa M. Nakayama K. Hatakeyama S. EMBO J. 2000; 19: 2069-2081Crossref PubMed Scopus (640) Google Scholar). Parental Skp2+/- mice used to generate Skp2+/+, Skp2+/-, and Skp2-/- littermates for the present study were derived by backcrossing the C57BL6/129SV knock-out strain onto the C57BL/6 background for seven or eight generations. We KKAy/TaJcl mice with Skp2+/- mice on the C57BL/6 background and then and Skp2+/- to and littermates on the C57BL/6 and mice were used for of the of a high fat mice were from of with fat by as described previously H. Ogawa W. M. S. Matsumoto M. K. N. Y. T. Sakaue H. K. S. Iguchi H. Hiramatsu R. D. K. Akira S. Kasuga M. Nat. Med. 2004; 10: Scholar). with mice were to the of the of of of and of adipose tissue for of cell size were obtained from the or fat The tissue was with as described J. E. J. Res. Full Text PDF PubMed Google Scholar), in and through a to The was then through a to adipocytes a of and the cells were with of cells was as described previously J. Res. 19: Full Text PDF PubMed Google Scholar) with the of a with a a and a CA). of the of adipocyte size was for cells from to in that the of the including is of WAT T. and M. the adipocyte number in WAT was by the WAT by the adipocyte The latter was by adipocyte A. J. Rev. PubMed Scopus Google Scholar) by adipocyte the latter of which was from the of adipocyte with the and was as described previously H. M. Ogawa W. T. T. M. M. H. S. Kasuga M. N. Genes Dev. 2002; PubMed Scopus Google Scholar, H. Ogawa W. Matsumoto M. S. M. T. Spiegelman B.M. Kasuga M. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). transcription and was also as described K. Ogawa W. Matsumoto M. Nakamura T. Sakaue H. Kasuga M. J. 2002; PubMed Scopus Google Scholar) with a and with mRNA as the The and were and for p27Kip1 and for Skp2 and and for cyclin E1 The for mRNA were as described previously K. Ogawa W. Matsumoto M. Nakamura T. Sakaue H. Kasuga M. J. 2002; PubMed Scopus Google Scholar). and and adipose tissue of mice were in in and at a of The were on and either with or to with to insulin and to were with or were with a The size of and that of β cells cell were by of at with was with an for of the were also with to insulin (Santa Cruz and and of were of for with glucose of for a glucose during which were at various An insulin was with mice in the glucose was at various of human insulin was as described previously C. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) with that of for were thus to of glucose the of insulin was at various and insulin was from the the and of glucose level was with a and insulin was with an and a insulin was with an for are as two were by as with of was of a on in the size and number of adipocytes in WAT of C57BL/6 mice a high fat diet or from of of mice on the high fat diet increased and the of WAT by and and by and 26 with mice the diet and The high fat diet also induced a marked increase in the size of adipocytes but effect on adipocyte number in WAT In 26 the high fat diet increased adipocyte size and number and The size of adipocytes not mice the high fat diet for or 26 These thus that an increase in adipocyte size an increase in adipocyte number in C57BL/6 mice a high fat The loss of p27Kip1 in mice in adipocyte hyperplasia as a result of increased proliferation or recruitment of preadipocytes (21Naaz A. Holsberger D.R. Iwamoto G.A. Nelson A. Kiyokawa H. Cooke P.S. FASEB J. 2004; 18: 1925-1927Crossref PubMed Scopus (121) Google Scholar). We the high fat diet p27Kip1 expression in of C57BL/6 mice on the high fat diet for which effect on adipocyte number, not the abundance of p27Kip1 in WAT, as with that in the diet In with the high fat diet for 26 resulted in a marked of the amount of p27Kip1 in WAT without an effect on the amount of the mRNA and Skp2 was not in WAT by that the amount of Skp2 mRNA in this tissue was increased in mice the high fat diet for 26 with that in and with to the of the high fat diet on p27Kip1 and Skp2 expression were obtained with WAT not of mice with the high fat diet for 26 also induced a in the abundance of cyclin of SCFSkp2 (23Pagano M. Benmaamar R. Cancer Cell. 2003; 4: 251-256Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), in WAT without a effect on the amount of the mRNA and The high fat diet thus increased the expression of Skp2 and that of p27Kip1 and cyclin E1 in WAT, and these were accompanied by an increase in adipocyte number. and β Cell in Skp2-/- the loss of Skp2 the of adipocytes to a high fat we WAT in Skp2-/- mice with previously described (25Nakayama K. Nagahama H. Minamishima Y.A. Matsumoto M. Nakamichi I. Kitagawa K. Shirane M. Tsunematsu R. Tsukiyama T. Ishida N. Kitagawa M. Nakayama K. Hatakeyama S. EMBO J. 2000; 19: 2069-2081Crossref PubMed Scopus (640) Google Scholar), Skp2-/- mice are their their was thus reduced by with that of at of The Skp2-/- mice at of were also as in marked in the of or WAT to the of WAT to and Skp2+/- mice. of adipocyte that adipocytes of WAT were in Skp2-/- mice in mice The number of adipocytes in WAT was reduced in Skp2-/- mice with that in mice and this thus to be for the in WAT of the of various or to in Skp2+/+, and mice at of not not in a were in the of adipose or to Skp2+/+, Skp2+/-, and Skp2-/- mice However, the of the of the to was in Skp2-/- mice in their of in or size and Skp2-/- and mice. In β cells were such as and were reduced in number in Skp2-/- mice and These mutant mice thus have reduced of adipocytes in WAT and of β cells in the with mice. in Skp2-/- glucose and insulin in the or not Skp2-/- and mice at of However, an glucose marked glucose in Skp2-/- mice An insulin that Skp2-/- and mice were to the effect of insulin further the of β cells in Skp2-/- mice, we the of insulin in to glucose C. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). The insulin of Skp2-/- mice was reduced with that of mice as as 2 of and this for to The the for the insulin and glucose was for Skp2-/- mice for their littermates These that the reduced number of β cells in Skp2-/- mice is in a reduced insulin to glucose and insulin of Skp2+/+, and mice high fat or fat not not in a of Skp2-/- to mice the high fat diet and in and and and of and Skp2+/- mice on this diet and from to and from to respectively. In Skp2-/- mice the high fat diet only from to and from to of The in the of WAT induced by the high fat diet was also for Skp2-/- mice for their littermates the size of adipocytes in WAT was for and Skp2-/- mice the high fat diet the number of these cells was reduced in the mutant suggesting that their was to this in adipocyte number. with the WAT mass in Skp2-/- mice with that in on the high fat the of was in the Skp2-deficient mice We the effect of the high fat diet on the abundance of p27Kip1 and cyclin E1 in WAT of Skp2-/- mice at of the high fat diet induced downregulation of p27Kip1 and cyclin E1 in WAT of mice, such in that of Skp2-/- mice The of p27Kip1 and cyclin E1 in WAT not mice of the two on the high fat diet These that degradation of p27Kip1 and cyclin E1 be for adipocyte proliferation in to a high fat of Skp2-/- a investigated the resistance of Skp2-/- mice to obesity induced by a high fat diet was accompanied by from insulin in glucose or insulin in the was to Skp2-/- and mice on the high fat diet and and and Skp2+/- not mice the high fat diet at of the insulin of Skp2-/- mice on this diet not and of in to of mice, of Skp2-/- mice increase in β cell mass in to the high fat diet these that the from obesity by Skp2 ablation was with of the induction of insulin resistance by a high fat an insulin that to insulin not Skp2-/- and mice such a diet of Skp2-/- to Obesity by the (Ay) that loss of Skp2 the development of obesity and insulin we generated Skp2-/- mice with the lethal yellow agouti (Ay) that this are as a well characterized of obesity. We thus Skp2+/- mice and mice, in which the Ay is present on the background H. A. Z. PubMed Scopus Google Scholar). We then obtained and littermates on the C57BL/6 and background by and Skp2+/- mice were found to be their littermates at of mice mice and of and in the two at of was at in part to a WAT in the The number of adipocytes in WAT was in mice in mice, adipocyte size not the two and glucose not and littermates in the at or of In mice, the of insulin was increased at and of with the development of obesity In mice, the of insulin at or of was to that at of An glucose glucose in Skp2-/- mice at of an insulin that these mice were to the effect of insulin at of of also in to that in mice, β cell mass was not increased in Skp2-/- mice at of The of WAT during the development of obesity is by an increase in the size of adipocytes and the of the latter of which is to as adipocyte We have that maintenance of C57BL/6 mice on a high fat diet for induced adipocyte without an increase in adipocyte number. In mice such a diet for an resulted in an increase in adipocyte number without a further increase in adipocyte The expansion of WAT mass in mice a high fat diet thus to be characterized by an initial increase in adipocyte size, a size is followed by an increase in adipocyte number. of mice on a high fat diet for 26 also resulted in of the expression of p27Kip1 in of p27Kip1 is in part by and degradation (23Pagano M. Benmaamar R. Cancer Cell. 2003; 4: 251-256Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). we were not to induction of Skp2 in WAT by the of p27Kip1 in WAT of mice the high fat diet was accompanied by an increase in the amount of Skp2 The high fat of p27Kip1 was also not in WAT of Skp2-/- mice. the of Skp2 protected mice on such a diet from the development of and this effect was to inhibition of adipocyte proliferation to of adipocyte hypertrophy. have a role for Skp2 in p27Kip1 degradation during the of adipocyte differentiation in 3T3-L1 preadipocytes in Biochem. Biophys. Res. Commun. 2006; PubMed Scopus Google Scholar, J. Cell. Biochem. 2006; Scholar). these suggest that degradation of p27Kip1 controls adipocyte proliferation during the development of obesity. In addition to a reduced number of adipocytes in WAT, Skp2-/- mice a high fat diet a reduced number of β cells in the We have previously that of p27Kip1 in β cells induced in mice as a result of inhibition of β cell proliferation T. Nakamura T. N. T. K. Sakaue H. Y. Y. Nakayama K.I. Kasuga M. Nat. Med. 2005; PubMed Scopus Google Scholar). The transcription factor which controls the G1 to S transition of the cell regulates the of the and the number of β but the size of β cells in E2F1 knock-out mice was found not to from that in L. S. D. M. Auwerx J. J. 2004; PubMed Scopus Google Scholar). The number and size of β cells were to be reduced in mice that for insulin and in β cells as well as in phosphoinositide-dependent kinase in these cells K. Okada T. J. A. M. I. R. M. Nat. 2006; PubMed Scopus Google Scholar, N. Y. T. S. Y. T. A. D. A. Ogawa W. Y. H. T. Kasuga M. 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Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), the reduced number of β cells and reduced level of insulin secretion in Skp2-/- mice to the inhibition of adipocyte proliferation during on a high fat is also that the reduced number of adipocytes a role in the reduction in β cell number. the of a in the number of adipocytes in WAT on insulin we an insulin in and Skp2-/- mice with the Ay of Skp2 in the Ay mutant mice resulted in marked of the effect of suggesting that the reduction in the number of adipocytes caused by Skp2 ablation in these inhibited the development of obesity-related insulin However, given that induces insulin resistance Y. Cell Biol. 2001; Full Text PDF PubMed Scopus Google Scholar) and in to the mice, the mice not of Skp2 knock-out mice be to this In the of with on a high fat diet in mice, and that with in in of the through an increase in adipocyte not have an for and long term of in an increase in adipocyte number to of the We have now that this increase in adipocyte number is with up-regulation of Skp2 expression in ablation of Skp2 resulted in inhibition of the increase in adipocyte number and of obesity-related insulin resistance in mice. We that Skp2 plays an essential role in adipocyte proliferation during the development of obesity. of the for the up-regulation of Skp2 expression in WAT during the development of obesity a for to obesity and obesity-related We N. and Y. for and respectively.