Báo cáo khoa học: Adipocyte hyperplasia and RMI1 in the treatment of obesity doc

MINIREVIEW Adipocyte hyperplasia and RMI1 in the treatment of obesity Akira Suwa, Takeshi Kurama and Teruhiko Shimokawa Drug Discovery Research, Astellas Pharma Inc., Ibaraki, Japan Introduction Obesity is a complex disorder and a major risk factor for metabolic diseases such as type 2 diabetes mellitus, hypertension and cardiovascular disease. Obesity devel- ops as the result of an imbalance between energy intake and expenditure. To state simply, chronic reduc- tion of energy expenditure versus intake causes an increased storage of the excess energy in the form of intracellular triacylglycerol droplets in adipose cells, leading to an increased fat mass and ultimately result- ing in obesity. Adipocyte hyperplasia (increase in cell number) and hypertrophy (increase in cell size) are thought to be directly responsible for the observed increase in adi- pose tissue mass [1,2]. Adipocyte hypertrophy in par- ticular is considered the main cause of adult obesity, and hyperplasia of adipocytes in obese adults some- times occurs secondary to adipocyte hypertrophy, pos- sibly due to an increased number of adipocytes capable of secreting paracrine growth factors that induce adipocyte hyperplasia [3]. Whereas the basic number of adipocytes is estab- lished during childhood and adolescence in both humans and rodents, adipose tissue retains the ability to generate new adipocytes throughout life. Increased adipocyte number during aging has been implicated in the rising incidence and severity of obesity among the elderly [4]. Thiazolidinediones, a class of oral antidia- betic agents, are nuclear receptor peroxisome prolifera- tor-activated receptor gamma agonists which enhance generation of small-sized adipocytes by inducing adi- Keywords adipocyte hyperplasia; adipogenesis; cell cycle; obesity; E2F; energy homeostasis; high-fat diet; metabolic disorders; RMI1; therapeutic target Correspondence A. Suwa, Pharmacology Research Labs, Drug Discovery Research, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba-shi, Ibaraki 305–8585, Japan Fax: +81 29 852 5391 Tel: +81 29 863 6417 E-mail: akira.suwa@jp.astellas.com (Received 26 July 2010, revised 29 November 2010, accepted 3 December 2010) doi:10.1111/j.1742-4658.2010.07980.x The escalating prevalence of obesity is one of the most pressing health concerns of the modern era, yet existing medicines to combat this global pandemic are disappointingly limited in terms of safety and effectiveness. The inadequacy of currently available therapies for obesity has made new drug development crucial. In the past several decades, however, major pro- gress has been achieved in understanding adipocyte hyperplasia associated with the pathogenesis of obesity, and consequently new potential targets for the medical treatment of obesity have been identified. We primarily review recent progress in the regulation of adipocyte hyperplasia as a novel emerging nontraditional approach. In this minireview, we focus on recQ- mediated genome instability 1 (RMI1), a recently identified novel molecular target for obesity treatment. RMI1-deficient mice have been found to be resistant to high-fat diet- and genetics-related obesity. Expression of this protein is regulated by E2F transcription factors, and recent studies have suggested that RMI1 plays an important role in the control of energy homeostasis during the development of obesity, with a mode of action based on the regulation of adipocyte hyperplasia. Abbreviations A y , lethal yellow agouti; BLM, Bloom’s syndrome gene; E2F, E2F transcription factor; RB, retinoblastoma protein; RMI1, recQ-mediated genome instability 1; Skp2, S-phase kinase-associated protein 2. FEBS Journal 278 (2011) 565–569 ª 2010 The Authors Journal compilation ª 2010 FEBS 565 pogenesis, resulting in increased adipose tissue mass and thereby obesity [5]. This growing body of evidence suggests that adipo- cyte hyperplasia may be a key event in the develop- ment and subsequent clinical course of some types of obesity. In this minireview, we focus on adipocyte hyperplasia as a novel therapeutic target for the treat- ment of obesity and discuss the merits of targeting recQ-mediated genome instability 1 (RMI1), a recently identified energy homeostasis-related molecule, for obesity treatment. Adipocyte hyperplasia Preclinical studies have demonstrated that adipocyte hyperplasia occurs in two steps: an increase in num- bers of preadipocytes, followed by the differentiation of preadipocytes into mature adipocytes. The transi- tion process from proliferation to differentiation in the adipocyte is tightly regulated by interaction between the cell-cycle regulators and the differentiating factors, and creates a cascade of events leading to the commit- ment of cells into the adipocyte phenotype [6,7]. This process, described as ‘adipogenesis’, requires a specific sequence of events to unfold, including growth arrest of proliferating preadipocytes, coordinated re-entry into the cell cycle with a limited clonal expan- sion, and growth arrest before terminal differentiation during lipid accumulation, suggesting that some cross-talk occurs between the cell cycle or the cell proliferation machinery and the factors controlling cell differentiation. Cell-cycle regulation The factors involved in cell-cycle regulation clearly fulfill important roles in the cell proliferative phase of adipocyte hyperplasia. Cell-cycle progression in mam- mals is governed at each phase of the cell cycle by various complexes of cell-cycle-related molecules, including cyclins, cyclin-dependent kinases, their inhib- itors and the retinoblastoma protein (RB), as well as E2Fs. E2F and RB family members appear to participate in the regulation of cell-cycle events that are required for adipogenesis. In growth-arrested preadipocytes, for example, E2Fs are complexed with pRB, leading to repression of its target genes [8]. Upon re-entry into the cell cycle by these growth-arrested preadipocytes, RB is phosphorylated by cyclin ⁄ cyclin-dependent kinase holoenzymes, releasing the E2F complex and thereby resulting in activation of the E2F target genes [9]. Fajas et al. [10] demonstrated that the E2F protein family plays a central role in preadipocyte proliferation and that E2F1-deficient mice are resistant to obesity induced by a high-fat diet (due to suppression of fat mass accumulation). Cyclin-dependent kinase inhibitors include two fami- lies of proteins, the Cip (Kip) family and Ink4 family, and are central players in the exit of cells from the cell cycle [11]. Loss of p27 Kip1 or p21 Cip1 in mice leads to adipocyte hyperplasia as a result of increased prolifera- tion or recruitment of preadipocytes [12], suggesting that these cyclin-dependent kinase inhibitors are important in the regulation of adipocyte number. The S-phase kinase-associated protein (Skp)1–Cullin- F-box protein (SCF) ubiquitin ligase (E3) complex targets cyclin-dependent kinase inhibitors for degrada- tion by the 26S proteasome and thereby regulates cell cycle progression [13]. Sakai et al. [14] showed that a deficiency in Skp2, the substrate-binding subunit of the SCF Skp2 complex, contributes to the degradation of p27 Kip1 , resulting in an induced resistance to obesity due to inhibition of preadipocyte proliferation without causing adipocyte hypertrophy. Such results were observed in both high-fat diet- and lethal yellow agouti (A y ) gene-induced obesity models. Similarly, other cell-cycle-related molecules such as cyclin-dependent kinase [15,16] and RB [17] have also been shown to play an important role in cell proliferation during adipogenesis. The above-mentioned findings strongly suggest that cell-cycle regulation associated with adipocyte hyper- plasia may present a novel approach to treating obes- ity. However, any cell-cycle modulation could potentially affect not only metabolic tissue, but normal tissues as well. Indeed, Skp2-deficient mice have shown a reduced number of b cells in the pancreas [14]. In addition, overexpression of p27 Kip1 in b cells induced hyperglycemia in mice as a result of inhibition of b-cell proliferation [18]. These potentially serious adverse effects associated with nonspecific inhibition of cell proliferation may pose a major obstacle for clinical use of this approach in obesity therapy. RMI1 and obesity Using a random mutagenesis approach based on the exchangeable gene trap method, we recently identified RMI1 as a novel regulator of energy homeostasis [19], which was reported to be an essential component of Bloom’s syndrome protein complexes [20], although no evidence had linked it to energy homeostasis until our findings. RMI1, an enzyme-binding protein, has previously been reported to mediate DNA recombina- tion, chromosome organization and biogenesis, as well Adipocyte hyperplasia and RMI1 A. Suwa et al. 566 FEBS Journal 278 (2011) 565–569 ª 2010 The Authors Journal compilation ª 2010 FEBS as to regulate cell-cycle checkpoint machinery [21]. RMI1 is also a member of Bloom’s syndrome gene (BLM)–topoisomerase complex; targeted mutations of BLM are developmentally delayed and die off by embryonic day 13.5 [22,23] and RMI1 homozygous knockout embryos die due to an unknown cause [19]. Bloom’s syndrome is a rare recessive genetic disorder characterized by dwarfism, telangiectatic erythema, immune deficiency and a predisposition towards devel- oping cancer [24,25]. RMI1 heterozygous knockout mice (RMI1+ ⁄ )) have a phenotype almost identical to that of the wild- type, although body weight and fasting-plasma glucose are significantly lower in wild-type mice. However, RMI1+ ⁄ ) mice possess a number of striking features that render them resistant to metabolic diseases. When fed a high-fat diet, the mutant mice were not only resistant to obesity, they showed improved glucose intolerance and reduced abdominal fat tissue mass. In addition, the mutants were also resistant to obesity induced by the A y gene. Of particular note is the fact that the deficient mice showed a rate of weight gain and amount of food intake equivalent to measurements taken under normal diet conditions. Taken together, these results indicate that RMI1-deficient mice can grow normally despite developing basal abnormalities, suggesting that the impact of RMI1 deficiency is sensi- tive and limited to developing obesity [19]. However, we cannot exclude the possibility that slight basal changes such as lower body weight and as yet unidenti- fied abnormalities can partially affect the energy balance. Future studies are needed to clarify whether RMI1-deficient mice exhibit a BLM-like phenotype. RMI1 and cell-cycle regulation The mode of action of RMI1 in the regulation of energy homeostasis might be based on the regulation of adipocyte hyperplasia. Studies have shown that siRNA knockdown of RMI1 resulted in suppression of cell proliferation [24]. However, RMI1 expression is upreg- ulated in the abdominal fat tissue of obese and diabetic mice, which show adipocyte hyperplasia, suggesting that RMI1 may be associated with the development of lipid accumulation in metabolic tissues, thereby leading to obesity [19]. We recently observed that treatment with glucose increased RMI1 expression in adipocyte- derived cell lines. Intriguingly, both mRNA levels of E2F5 and E2F8 increased on treatment with glucose, as seen with RMI1, and siRNA knockdown of these genes suppressed RMI1 expression. In addition, com- puter analysis has shown evidence of E2F response ele- ment consensus sites in the RMI1 promoter (Suwa A, Kurama T, Shimokawa T and Aramori I, unpublished observations), suggesting that E2F may transactivate the promoter. Taken together, these results indicate that RMI1 expression can be regulated by E2F family molecules in adipose tissue under high-glucose condi- tions, influencing preadipocyte proliferation. RMI1 as a novel target for obesity treatment Recent studies in vivo and in vitro have demonstrated that RMI1 plays an important role in the regulation of energy homeostasis via an interaction with E2F path- ways, at least in part, suggesting the involvement in the regulation of adipocyte hyperplasia. RMI1 expres- sion is induced by glucose in vitro, and its in vivo expression is also induced by metabolic abnormalities such as hyperglycemia and obesity in metabolic tissues such as liver and adipose tissues. However, targeting RMI1 may not affect whole-body proliferation, because changes in RMI1 expression in metabolic disorders are restricted to metabolic tissues; indeed, RMI1-deficient mice have not shown any abnormalities in nonmetabolic tissues. More impor- tantly, the in vivo evidence of anti-obesity effects was obtained from RMI1 heterozygous mice, suggesting that a 50% reduction in RMI1 level would be sufficient to treat energy homeostasis disorders. Recent advanced findings about RMI1 suggest that RMI1-targeting is a helpful therapeutic approach to treating energy homeo- stasis disorders such as obesity, leading to an encourag- ing increase in research focusing on drug discovery of RMI1 expression modulators. However, several unan- swered questions remain regarding the effects of RMI1 on the regulation of energy homeostasis. First, RMI1 deficiency suppresses high-fat diet-induced upregulation of E2F8 in vivo [19]. By contrast, RMI1 expression is actually regulated by E2F8 expression in vitro,as described above. These reports permit us to speculate regarding the positive feedback regulation between RMI1 and E2F on cell proliferation. In addition, E2F8 has been reported to reduce rather than induce cell pro- liferation [26,27]. Given the disparity in findings, eluci- dation of E2F8’s functions and contribution to the regulation of cell proliferation will require numerous future experiments. Second, RMI1 deficiency signifi- cantly reduced food intake only under conditions of excess of energy input [19]. In addition, RMI1 expres- sion was significantly increased in the hypothalamus under high-fat feeding conditions and decreased under fasting conditions [19]. These results suggest the possi- bility that RMI1 might directly regulate feeding behav- ior via the central nervous system, which has crucial A. Suwa et al. Adipocyte hyperplasia and RMI1 FEBS Journal 278 (2011) 565–569 ª 2010 The Authors Journal compilation ª 2010 FEBS 567 roles in the control of energy metabolism, leading to body weight reduction. Further investigations to clarify the each contribution of adipocyte and central effects on the regulation of energy homeostasis such as using the brain- or adipose tissue-specific RMI1 conditional knockout mice would be required. Conclusions Obesity develops as a result of the disruption of the homeostasis between food intake and energy expendi- ture, and therefore factors affecting these processes are the focus of extensive research targeting the develop- ment of effective anti-obesity drugs. To date, however, only limited success has been achieved in this endeavor [28], highlighting the need for additional therapeutic options. Recently, interesting novel approaches and tar- gets for obesity treatment have been reported, many of which have been cited in the other minireviews in this series. One such approach is the concept of central regu- lation, focusing on the malonyl-CoA pathway [29], while another approach uses the adipocyte-derived inflammatory mediator [30]. However, here we focus on a recently identified novel approach involving adipocyte hyperplasia, differing clearly from both the central and inflammatory approaches. Given that a number of molecular causes have been implicated in the develop- ment of obesity, combining these different approaches likely represents the most effective treatment method. Excessive calorie intake associated with hyperphagia and ingesting a high-fat diet in mice results in storage of the extra energy, initially through an increase in adi- pocyte size. However, as adipocytes have a limited capacity for enlargement, long-term intake of excess calories eventually results in an increase in adipocyte number to accommodate storage of the surplus energy. In this minireview, we have summarized evidence regarding the regulation of adipocyte number as a novel promising approach to treating obesity, and this approach may represent a target for novel anti-obesity drugs that prevent adipocyte hyperplasia. Any thera- peutic potential, however, is not well established at present, and therefore more detailed understanding of adipocyte hyperplasia in humans is essential for the development of efficacious and safe treatments. As emphasized here, RMI1 is one of the most promis- ing targets for obesity treatment with the potential to strongly influence care of obesity patients. Our hypothetical model for the regulation of adipo- cyte hyperplasia by RMI1 is shown in Fig. 1. Excess calorie intake induces expression of RMI1 and E2Fs in adipocytes, and these changes in expression are expected to increase a positive interaction between the molecules. The increased expression of RMI1 and E2Fs thereby regulates the cell cycle, leading to adipocyte hyperplasia and the development of obesity. Further studies are clearly needed to explore the detailed mechanism and interaction between RMI1 and E2F molecules, as well as to determine the clinical relevance of RMI1. Acknowledgements We thank Drs Masao Kato, Ichiro Aramori, Hitoshi Matsushime, Masato Kobori, Jiro Hirosumi, Masa- yasu Yoshino, Shun-ichiro Matsumoto, and Masanori Naitou at Astellas Pharma Inc., and Mses Chihiro Yamazaki and Rie Fujikawa at Trans Genic Inc. for their helpful advice and support. References 1 Hausman DB, DiGirolamo M, Bartness TJ, Hausman GJ & Martin RJ (2001) The biology of white adipocyte proliferation. Obes Rev 2, 239–254. RMI1 RMI1 The intake of excess calories (Hyperphagia, high-fat feeding) E2F E2F Obesity Obesity (Energy homeostasis disorder) (Energy homeostasis disorder) Adipocyte hyperplasia Cell cycle regulation Expression Expression regulation regulation Fig. 1. A hypothetical model for adipocyte hyperplasia regulation by RMI1. Excess calorie intake due to hyperphagia and high-fat feed- ing induces expression of RMI1 and E2Fs in adipocytes, and causes a positive interaction between the molecules. Increased expression of RMI1 and E2Fs regulates the cell cycle, leading to adipocyte hyperplasia and subsequent development of obesity. Adipocyte hyperplasia and RMI1 A. Suwa et al. 568 FEBS Journal 278 (2011) 565–569 ª 2010 The Authors Journal compilation ª 2010 FEBS 2 Avram MM, Avram AS & James WD (2007) Subcuta- neous fat in normal and diseased states: 3. Adipogene- sis: from stem cell to fat cell. J Am Acad Dermatol 56, 472–492. 3 Marques B, Hausman D & Martin R (1998) Associa- tion of fat cell size and paracrine growth factors in development of hyperplastic obesity. Am J Physiol 275, R1898–R1908. 4 Kirkland J, Hollenberg C & Gillon W (1990) Age, anatomic site, and the replication and differentiation of adipocyte precursors. Am J Physiol 258, C206–C210. 5 Arner P (2003) The adipocyte in insulin resistance: key molecules and the impact of the thiazolidinediones. Trends Endocrinol Metab 14, 137–145. 6 Gregoire F, Smas C & Sul H (1998) Understanding adipocyte differentiation. Physiol Rev 78, 783–809. 7 Rosen E & Spiegelman B (2000) Molecular regulation of adipogenesis. Annu Rev Cell Dev Biol 16, 145–171. 8 Dyson N (1998) The regulation of E2F by pRB-family proteins. Genes Dev 12, 2245–2262. 9 Richon V, Lyle R & McGehee RJ (1997) Regulation and expression of retinoblastoma proteins p107 and p130 during 3T3-L1 adipocyte differentiation. J Biol Chem 272, 10117–10124. 10 Fajas L, Landsberg RL, Huss-Garcia Y, Sardet C, Lees JA & Auwerx J (2002) E2Fs regulate adipocyte differen- tiation. Dev Cell 3, 39–49. 11 Nakayama K & Nakayama K (2006) Ubiquitin ligases: cell-cycle control and cancer. Nat Rev Cancer 6, 369–381. 12 Naaz A, Holsberger D, Iwamoto G, Nelson A, Kiyo- kawa H & Cooke P (2004) Loss of cyclin-dependent kinase inhibitors produces adipocyte hyperplasia and obesity. FASEB J 18, 1925–1927. 13 Zhang H, Kobayashi R, Galaktionov K & Beach D (1995) p19Skp1 and p45Skp2 are essential elements of the cyclin A–CDK2 S phase kinase. Cell 82, 915–925. 14 Sakai T, Sakaue H, Nakamura T, Okada M, Matsuki Y, Watanabe E, Hiramatsu R, Nakayama K, Nakay- ama KI & Kasuga M (2007) Skp2 controls adipocyte proliferation during the development of obesity. J Biol Chem 282, 2038–2046. 15 Tang Q-Q, Otto T & Lane MD (2003) CCAAT ⁄ enhancer-binding protein beta is required for mitotic clonal expansion during adipogenesis. Proc Natl Acad Sci USA 100, 850–855. 16 Abella A, Dubus P, Malumbres M, Rane SG, Kiyokawa H, Sicard A, Vignon F, Langin D, Barb- acid M & Fajas L (2005) Cdk4 promotes adipogenesis through PPAR[gamma] activation. Cell Metab 2, 239– 249. 17 Hansen J, Petersen R, Jørgensen C & Kristiansen K (2002) Deregulated MAPK activity prevents adipocyte differentiation of fibroblasts lacking the retinoblastoma protein. J Biol Chem 277, 26335–26339. 18 Uchida T, Nakamura T, Hashimoto N, Matsuda T, Kotani K, Sakaue H, Kido Y, Hayashi Y, Nakayama K, White M et al. (2005) Deletion of Cdkn1b ameliorates hyperglycemia by maintaining compensatory hyperinsulinemia in diabetic mice. Nat Med 11, 175–182. 19 Suwa A, Yoshino M, Yamazaki C, Naitou M, Fujikawa R, Matsumoto S, Kurama T, Shimokawa T & Aramori I (2010) RMI1 deficiency in mice protects from diet and genetic-induced obesity. FEBS J 277, 677–686. 20 Wu L, Bachrati CZ, Ou J, Xu C, Yin J, Chang M, Wang W, Li L, Brown GW & Hickson ID (2006) BLAP75 ⁄ RMI1 promotes the BLM-dependent dissolu- tion of homologous recombination intermediates. Proc Natl Acad Sci USA 103, 4068–4073. 21 Mankouri HW & Hickson ID (2007) The RecQ helicase-topoisomerase III–Rmi1 complex: a DNA structure-specific ‘dissolvasome’? Trends Biochem Sci 32, 538–546. 22 Chester NKF, Kozak C, O’Hara CD & Leder P (1998) Stage-specific apoptosis, developmental delay, and embryonic lethality in mice homozygous for a targeted disruption in the murine Bloom’s syndrome gene. Genes Dev 12, 3382–3393. 23 Luo GSI, McDaniel LD, Nishijima I, Mills M, Yous- soufian H, Vogel H, Schultz RA & Bradley A (2000) Cancer predisposition caused by elevated mitotic recom- bination in Bloom mice. Nat Genet 26, 424–429. 24 Yin JSA, Xu C, Meetei AR, Hoatlin M, Li L & Wang W (2005) BLAP75, an essential component of Bloom’s syndrome protein complexes that maintain genome integrity. EMBO J 24, 1465–1476. 25 German J (1995) Bloom’s syndrome. Dermatol Clin 13, 7–18. 26 Christensen J, Cloos P, Toftegaard U, Klinkenberg D, Bracken AP, Trinh E, Heeran M, Di Stefano L & Helin K (2005) Characterization of E2F8, a novel E2F-like cell-cycle regulated repressor of E2F-activated transcrip- tion. Nucleic Acids Res 33, 5458–5470. 27 Maiti B, Li J, de Bruin A, Gordon F, Timmers C, Opavsky R, Patil K, Tuttle J, Cleghorn W & Leone G (2005) Cloning and characterization of mouse E2F8, a novel mammalian E2F family member capable of block- ing cellular proliferation. J Biol Chem 280, 18211– 18220. 28 Gura T (2003) Obesity drug pipeline not so fat. Science 299, 849–852. 29 Wolfgang M & Lane MD (2010) Hypothalamic malo- nyl-CoA and CPT1c in the treatment of obesity. FEBS J 278, 552–558. 30 Kadomatsu T, Tabata M & Oike Y (2010) Angiopoie- tin-like proteins: emerging targets for the treatment of obesity and related metabolic diseases. FEBS J 278, 559–564. A. Suwa et al. Adipocyte hyperplasia and RMI1 FEBS Journal 278 (2011) 565–569 ª 2010 The Authors Journal compilation ª 2010 FEBS 569 . positive interaction between the molecules. The increased expression of RMI1 and E2Fs thereby regulates the cell cycle, leading to adipocyte hyperplasia and the. recruitment of preadipocytes [12], suggesting that these cyclin-dependent kinase inhibitors are important in the regulation of adipocyte number. The S-phase kinase-associated