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

Thiazolidinediones, a class of oral antidia-betic agents, are nuclear receptor peroxisome prolifera-tor-activated receptor gamma agonists which enhance generation of small-sized adipocyt

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

Ay, 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.

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 proliferatransi-tion to differentiatransi-tion 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 p27Kip1or p21Cip1 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 SCFSkp2 complex, contributes to the degradation of p27Kip1, 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 (Ay) 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 p27Kip1 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

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 Ay 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 focusencourag-ing 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

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 adiadi-pocytes 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

The intake of excess calories

(Hyperphagia, high-fat feeding)

E2F

Obesity

(Energy homeostasis disorder)

Adipocyte hyperplasia

Cell cycle regulation

Expression 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.

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 recombinadissolu-tion 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