The tumor suppressors pRB and p53 as regulators of adipocyte differentiation and function

Review

The tumor suppressors pRB and p53 as regulators of adipocyte differentiation and function Philip Hallenborg , Søren Feddersen , Lise Madsen & Karsten Kristiansen † † University of Southern Denmark, Department of Biochemistry and Molecular Biology, Campusvej 55, 5230 Odense M, Denmark

Background : The retinoblastoma protein (pRB) and p53 are crucial members of regulatory networks controlling the cell cycle and apoptosis, and a hallmark of virtually all cancers is dysregulation of expression or function of pRB or p53. Although they are best known for their role in cancer development, it is now evident that both are implicated in metabolism and cellular development. Objective/methods : To review the role of pRB and p53 in adipocyte differentiation and function emphasizing that pRB and p53, via their effects on adipocyte development and function, play a role in the regulation of energy metabolism and homeostasis. Results/conclusions : pRB is required for adipose conversion and also involved in determining its mitochondrial capacity. p53 inhibits adipogenesis and results suggest that it is involved in maintaining function of adipose tissue.

Keywords: brown adipose tissue , obesity , p53 , pRB , UCP1 , white adipose tissue

Expert Opin. Ther. Targets (2009) 13 (2):235-246

1. Introduction

An epidemic-like rise in the incidence of obesity has been observed worldwide during the last decades. The excessive accumulation of adipose tissue characterizing obesity is accompanied by a state of chronic inflammation in- and dysfunction of- adipose tissues. Proper function of the adipose tissue is crucial for whole body homeostasis due to its role in storage and release of energy and secretion of hormones [1-3] .

Obesity places an enormous economic burden on society as a number of diseases are found in its path, especially type 2 diabetes and cardiovascular disorders. Furthermore, it is well described that certain cancers are also associated with obesity [4,5] . It has been estimated that in the United States obesity could account for as much as 14% of all deaths from cancer in men and 20% of them in women [6] .

Several genes are known to be involved in cell cycle control and tumorigenesis. Two of these, the retinoblastoma protein (pRB) and p53, are well known tumor suppressors and stand today as some of the best described genes. Although their roles in regulating the cell cycle are well characterized, their involvement in cellular differentiation and metabolism is receiving more and more attention. Here we review their role in adipocyte differentiation and function.

1.1 The retinoblastoma protein, pRB The intense study of the molecular mechanisms controlling formation of retinoblastoma tumors led to the cloning of the RB1 gene [7] . Since then, the gene has been reported to be inactivated in several cancer types [8] . The product of the gene, pRB, functions as a cofactor for many transcription factors, but is

1. Introduction

2. Adipocyte differentiation

3. Adipocyte function

4. Conclusion

5. Expert opinion

The tumor suppressors pRB and p53 as regulators of adipocyte differentiation and function

236 Expert Opin. Ther. Targets (2009) 13(2)

best known for its importance in regulating the activity of the E2Fs. pRB is vital for controlling the cell cycle by blocking exit from G0 to G1 and progression through G1 into S phase. The importance of pRB in regulating cell cycle progression is underscored not only by the frequent inactivation of pRB in human cancers but is also apparent during embryogenesis where pRB-deficient embryos die due to a dysfunctional placenta caused by excessive trophoblast proliferation [9] . Besides its unquestionable role in regulating the cell cycle, pRB is also involved in differentiation of several cell types. For example, proper erythropoiesis and myogenesis require pRB [10,11] .

pRB is the founding member of the pocket protein family comprising pRB, p107 and p130. Like pRB, the close relatives p107 and p130 are also involved in cell cycle control. Curiously, the phenotypes of mice deficient for either p107 or p130 depend on the strain of mice used [12] .

1.2 p53 The gene encoding the transcription factor p53 is one of the ones, if not the one, most frequently mutated gene in cancer. The importance of p53 in controlling tumor formation is emphasized by the broad spectrum of tumors found in mice lacking p53 [13,14] . As p53 originally was linked to cell cycle arrest or apoptosis upon DNA damage, it is also often referred to as ‘Guardian of the Genome’. Numerous studies have since shown that several other types of cellular stresses induce the activity of p53 leading to expression of a variety of genes involved in either blocking cell cycle progression or removal of the damaged cell through apoptosis [15,16] .

p53 is not unique. Two close relatives, p63 and p73, have been cloned and characterized. In contrast to the normal development of p53-deficient mice which have no gross developmental phenotype, mice lacking either p63 or p73 suffer from severe disorders [17-19] . It has been speculated that the absence of gross developmental defects in p53-deficient mice is due to a redundant function of one of the other family members [20] as the ablation of p53 in Xenopus, which lack p63 and p73, results in defective embryonic development [21] .

2. Adipocyte differentiation

The intake of a surplus of energy results in the storage of excess energy as fat in adipose depots found in many locations in the body. The main constituent of the adipose tissue is the adipocytes. However, appearance and function vary significantly between the different depots. Not all depots are even involved in energy storage and release. Adipose tissues around joints are mainly used for support and are not believed to take part in energy homeostasis. Adipocytes have traditionally been divided according to colors: brown and white. The two types handle excess energy in opposite ways. In contrast to the classic white adipocyte

which mainly functions as storage for excess energy, brown adipocytes convert surplus energy into heat.

The development of adipocytes is normally considered to take place in two segregated steps: the determination to eventually become an adipocyte and the differentiation of the dormant preadipocyte into a fat-laden, mature adipocyte. Adipocytes originate from mesenchymal stem cells (MSC). Other descendants include osteocytes, chondrocyte and myocytes. It has hitherto been assumed that each cell type is derived from special lineages of MSCs determined to undergo a certain differentiation program. This nearly dogmatic view was recently challenged by the discovery that brown adipocytes originate from MSCs with myogenic characteristics [22,23] . The later separation into either a myocyte or brown adipocyte relies on the transcriptional cofactor positive-regulatory-domain-containing 16 (PRDM16) [23] .

Like adipocytes, myocytes can also roughly be separated into two groups. Muscles are generally distinguished based on their fiber type: slow-twitch and fast-twitch muscles. In contrast to fast-twitch muscles, their slow counterparts are suited for prolonged exercise with the high oxidative capacity. Based on the myogenic signature in brown preadipocytes, it is tempting to speculate that the two myocyte types may originate from a different premyogenic cell lineage and that early determination reflects the oxidative capacity of the final cell such that slow-fiber type myocytes and brown adipocytes are descendants from one lineage, and the fast-fiber type myocytes and white adipocytes originate from different distinct lineages of MSCs. Conversion between the different types of myocytes may occur similarly to the transdifferentiation of white adipose tissue (WAT) into brown adipose tissue (BAT) that can be observed in response to β -adrenergic stimulation (see later).

Much is still to be learned about the determination of cell fate and the origins of myocytes and adipocytes. The differentiation of preadipocytes into mature adipocytes is far better described. The generally accepted dogma focuses on an early priming of adipogenesis by CCAAT/enhancer binding protein (C/EBP) β and δ followed by induction of peroxisome proliferator-activated receptor γ (PPAR γ ) and C/EBP α , which are responsible for the final development of the mature, insulin-responsive adipocyte [24] . The three C/EBPs and PPAR γ are thought to constitute the central axis of adipogenesis.

Several genes are reported to affect the mitochondrial activity of the adipocyte and have the ability to turn a white adipocyte into a brown-like adipocyte (see later and [25] ). Few genes, however, have been shown to be involved in the differentiation of only one of the adipocyte types. Interestingly, a member of the central axis appears to be important solely for white adipogenesis. The absence of WAT but presence of BAT in mice lacking C/EBP α in all tissues but the liver underscores the importance for this transcription factor in white adipogenesis [26] . A group of genes has been shown to exhibit selectivity in white versus

Hallenborg, Feddersen, Madsen & Kristiansen

Expert Opin. Ther. Targets (2009) 13(2) 237

brown adipogenesis. Bone-morphogenetic protein 7 (BMP7) has been shown to promote differentiation of brown preadipocytes but did not affect the adipose conversion of preadipocytes originating from WAT [27] . Two other BMPs, BMP2 and 4, have previously been shown to augment white adipogenesis [28,29] . The stimulatory effect is selective for white adipogenesis as both BMPs are able to suppress uncoupling protein 1 (UCP1) expression in differentiating brown preadipocytes [27] .

2.1 pRB in adipocyte differentiation The importance of pRB in adipocyte differentiation is illustrated by the inability of pRB-deficient fibroblasts to undergo adipose conversion [30,31] . Interestingly, much of the adipogenic effect of pRBs has been ascribed to its regulation of C/EBPs, C/EBP β in particular. pRB has been reported to bind to and augment the activity of C/EBP β during early adipose conversion [30] and although an inhibitory effect of pRB on C/EBP β has been reported [32] , mouse embryonic fibroblasts (MEFs) lacking pRB share some of the same characteristics as cells with ablation or decreased activity of C/EBP β . Compared with wild type cells, fewer pRB-deficient MEFs undergo mitotic clonal expansion [31] which at least in part is controlled by C/EBP β [33] . Induction of xanthine oxidoreductase (XOR), whose expression is coupled to C/EBP β activity [34] , is blunted in MEFs lacking pRB (Hallenborg et al ., unpublished data). Finally, adipogenesis is restored by a potent PPAR γ ligand both in pRB-deficient MEFs [31] and in preadipocytes with forced expression of a dominant negative C/EBP β [35] .

As pRB has been reported to regulate the activity of several transcription factors, it is likely that its adipogenic effect is not solely attributable to its regulation of C/EBP β activity. For instance, two E2Fs, the classic targets for pRB, are also reported to affect adipocyte differentiation. The report of a positive effect of E2F1 on the PPAR γ 1 promoter [36] , and thereby adipogenesis, contradicted an earlier observation showing an inhibition of adipose conver-sion by forced expression of E2F1 [37] . We therefore favor a model where a transient increase in E2F1 activity promotes adipogenesis whereas prolonged activity abrogates differentia-tion. pRB or its family members are then thought to partake in this fine-tuning of E2F1 activity. In contrast to the divergent results concerning the role of E2F1, there is general agreement that E2F4 functions as a repressor of adipocyte development [36,38] . Based on the central role of pRB and its family members in regulating the activity of the E2Fs, it is conceivable that the pocket proteins control this family of transcription factors during adipocyte differentia-tion in addition to the role of pRB as a regulator of the function of C/EBPs.

In summary, available data suggest that the requirement for pRB in adipogenesis relies on its ability to bind and augment the activity of C/EBP β although pRBs classic

targets, the E2Fs, are also likely to be under strict pRB control during adipose conversion.

2.2 p53 in adipocyte differentiation Even though the effects of p53 on differentiation of several cell types have been examined [39] , no reports have to date examined the possible role of p53 in adipocyte differentia-tion. Both stromal cells from the bone marrow and cells from the stromal-vascular fraction of BAT from mice lack-ing p53 have the ability to undergo adipose conversion [40,41] showing that p53 is not required for adipogenesis. Rather, we have observed an inhibitory effect of ectopic expression of p53 on adipogenesis of MEFs and an increased propensity of p53 -/- MEFs to undergo adipogenesis even in the absence of the hormonal induction normally required for adipogenesis. The inhibitory effect of p53 is dependent on an intact transactivating function, as forced expression of a transcrip-tionally inactive p53 fail to decrease adipose conversion. Activated p53 induces either cell cycle arrest or apoptosis and since cells with forced expression of p53 do not die it is plausible that it is the ability to block cell division that gives p53 its antiadipogenic effect. This is supported by the ability of roscovitine, a cyclin-dependent kinase (CDK) inhibitor, to block the spontaneous adipogenesis in MEFs lacking p53 (Hallenborg et al ., unpublished data).

Conversion of murine preadipocytes into mature adipo-cytes involves two normally mutually exclusive processes: cell division and terminal differentiation. As delineated by in vitro studies, the process of adipocyte differentiation of murine preadipocytes involves two rounds of cell division referred to as clonal expansion of the otherwise growth-arrested confluent cells. This step is suggested to be a prerequisite for adipogenesis of 3T3-L1 preadipocytes [42] . Interestingly, entry into S-phase is sufficient for adipose conversion of C3H10T1/2 [43] . The presence of a PPAR γ -ligand greatly augments adipogenesis of C3H10T1/2 which is in sharp contrast to 3T3-L1 preadipocytes where almost all cells undergo adipose conversion without addition of an exogenous PPAR γ ligand [44] . Human fibroblasts with the ability to differentiate into adipocytes do not undergo mitotic clonal expansion. They do however require addition of a PPAR γ ligand in order to differentiate [45] . One way of interpreting these results is that the clonal expansion is linked to PPAR γ ligand production.

Several lines of evidence link clonal expansion to the production of PPAR γ ligand(s) during adipogenesis. First, the two processes take place within the same timeframe during the differentiation program [42,46] . Second, the block in differentiation seen in 3T3-L1 preadipocytes with ablated C/EBP β is restored by administration of an exogenous PPAR γ ligand [35] , and C/EBP β has been shown to be required for clonal expansion and PPAR γ -ligand production [33-35] . Third, the block in adipose conversion of MEFs both by forced expression of p53 and the inhibitory effect on adipogenesis of several cell cycle inhibitors can be circumvented by an

exogenous PPAR γ ligand. Lastly, these cell cycle inhibitors decreased the activity of a PPAR γ -ligand-sensitive reporter system during differentiation of 3T3-L1 preadipocytes (Hallenborg et al ., unpublished data). Thus, it is tempting to hypothesize that clonal expansion is a prerequisite for PPAR γ ligand production and that the process of clonal expansion eliminates the need for exogenous ligand(s).

A possible scenario for the involvement of clonal expansion in PPAR γ ligand production is depicted in Figure 1 . Upon entering the S-phase, C/EBP β acquires DNA-binding activity [32,42] . C/EBP β then induces the expression of XOR. XOR is normally linked to purine catabolism but is also involved in production of reactive oxygen species (ROS). These ROS are highly reactive and they are especially effective in oxidizing lipids. As cells undergo cell division the production of phospholipids increase dramatically and it is likely that significant amounts of polyunsaturated fatty acids (PUFAs) are released during the extensive membrane remodeling as cells enter mitosis. PPAR γ is activated by a plethora of unsaturated, oxidized lipid [47] which could be generated by ROS-mediated attack on the released PUFAs.

Collectively, p53 stands as a negative regulator of adipo-genesis through its ability to block cell cycle progression which in the case of adipocyte differentiation seems to block PPAR γ ligand production.

3. Adipocyte function

Adipose tissue comes in two major types, white and brown, that have opposite functions in whole body energy homeo-stasis. WAT is the primary site of energy storage but is also considered to be the largest endocrine organ in mammals. Adipocytes secrete a large number of cytokines and hormones that act in an autocrine, paracrine and endocrine manner and regulate whole body metabolism. Thus lipodystrophy causes hormonal, reproductive and developmental abnormalities, insulin resistance and diabetes. Indeed, white adipose tissue plays an important role in whole-body glucose homeostasis. Diabetes may be reversed by leptin in models with moderate fat deficiency [48] , but

C/EBPβ

p53Clonal

expansionPUFA Ligand

PPARγ

Figure 1 . Pathway for generation of peroxisome proliferator-activated receptor g (PPAR g ) ligands through CCAAT/enhancer binding protein- b (C/EBP b )-induced xanthine oxidoreductase (XOR) expression and mitotic clonal expansion. PUFA: Polyunsaturated fatty acid; ROS: Reactive oxygen species.

not in severe models of lipoatrophy [49] . Even though skeletal muscle is the major tissue for insulin-stimulated uptake of glucose, the uptake of glucose in adipose tissue is significant and important; a fact underscored by the finding that adipose-specific glucose transporter 4 (GLUT4) knockout mice develop glucose intolerance [50] and adipose-specific overexpression of GLUT4 reverses diabetes in muscle-specific GLUT4 knockout mice [51] . Increased adipose tissue mass, on the other hand, also leads to glucose intolerance and insulin resistance. Obesity is accompanied by increased levels of cytokines and chemokines secreted from adipose tissue and a variety of these are believed to contribute to development of obesity-related disorders, type 2 diabetes and cardiovascular diseases in particular [3,52] . Whereas white adipocytes store excess energy as fat, brown adipocytes contain a large number of mitochondria dedicated to dissipate energy in form of heat through uncoupled respiration as a result of expression of the uncoupling protein UCP1. Thus, an increased abundance of brown adipocytes expressing UCP1 can counteract diet-induced obesity. Increased UCP1 expression as observed after transgenic expression of UCP1 itself [53,54] , forkhead box protein c2 (Foxc2) [55] , or disruption of genes encoding initiation factor 4E binding protein 1 (4E-BP) [56,57] , Cidea [58] , or pRB [59] protects mice from diet-induced obesity.

Brown adipocyes are mainly found in the brown-colored fat pad located in the intrascapular region. However, several reports indicate that brown-like multilocular adipocytes that express UCP1 are interspersed within the white adipose depots in both rodents [60,61] and humans [62-64] . It is not yet clear if the brown-like adipocytes found in typically WAT are identical or similar to the brown adipo-cytes located in the intrascapular region. However, several lines of evidence suggest that these brown-like adipocytes originate by transformation of mature white adipocytes [65,66] . First, in a cold environment, the number of brown-like adipocytes increases in certain white adipose tissues without a concomitant increase in the number of adipocytes and DNA content [66,67] . Also brown adipocytes located in the intrascapular region but not brown-like adipocytes appear-ing in WAT in response to β -adrenergic agonist arise from myogenic factor 5 (Myf5)-expressing progenitors [23] . Since the brown-like adipocytes found in WAT depots express UCP1, they may dissipate energy in the form of heat. Indeed, mouse strains that have more UCP1-expressing adipocytes in their white adipose tissue depots are protected against diet-induced obesity [61,68] .

3.1 pRB in adipocyte function A very interesting feature of pRB-deficient adipocytes is their high level of UCP1 expression. MEFs deficient for pRB differentiated in the presence of the PPAR γ ligand rosiglitazone express UCP1 at a level comparable to that of BAT [69] . In many ways pRB-deficient adipocytes resemble brown adipocytes. They have an increased number

of mitochondria compared with wild-type adipocytes [69] . They have increased or decreased expression of genes epreviously shown to be either BAT or WAT-specific, respectively. However, expression of certain genes normally considered WAT-selective such as Serpin3ak [70] is increased in pRB-deficient adipocytes ( Figure 2 ). Interestingly, before induction of myogenesis cells lacking pRB have increased expression of myogenin and myosin heavy chain compared with wild-type cells [71] indicating that fibroblasts lacking

pRB have increased susceptibility to entering the myocyte/brown adipocyte lineage. We therefore suggest a model as depicted in Figure 3 , where pRB favors the entry of a stem cell into the white adipocyte lineage.

The brown-like adipose phenotype was to some extent recapitulated in an in vivo study with adipose specific ablation of RB. These mice (RB ad-/- ) had increased expression of UCP1 in their white adipose depots [59] . When they were fed a high-fat diet (HFD) for 6 weeks the WAT and the

PGC-1α

PRDM16

Serpin3ak

Resistin

WAT-specific BAT-specific

Figure 2 . Expression of white and brown adipose markers in wild type and Rb -/- adipocytes. Mouse embryonic fi broblasts were differentiated as described previously [31] . cDNA synthesis and real-time PCR analyses were performed as described elsewhere [98] . Primer sequences have been described by Seale et al . [70] . Expressions are given relative to TBP. BAT: Brown adipose tissue; PGC-1 α : PPAR γ -cofactor 1 α ; PRDM16: Positive-regulatory-domain-containing 16; Psat1: Phosphoserine aminotransferase 1; TBP: TATA-box binding protein; UCP1: Uncoupling protein 1; WAT: White adipose tissue.

BAT of these mice contained an increased number of mitochondria and smaller lipid droplets compared with control mice. Moreover, under HFD RB ad-/- mice had higher O 2 consumption, produced more CO 2 , and exhibited increased thermogenesis. Collectively, these observations strongly indicate that increased energy expenditure protects RB ad-/- mice against obesity and hepatosteatosis. As the mice had the RB gene excised relatively late in the adipo-genic program (excision of RB was controlled by the promoter of the gene encoding the late adipocyte marker, adipocyte protein 2 (aP2)), this study shows that lack of pRB not only favors a more brown-like adipocyte phenotype by acting early during adipogenesis, but may also promote a transition towards a brown-like phenotype during later stages of adipo-cyte differentiation. Still, the adipose tissue of mice with specific knockout of RB in the adipose tissue failed to fully recapitulate the brown adipose phenotype in WAT [59] . It is therefore conceivable that pRB acts on several levels to control BAT-development and/or UCP1 expression.

Several mechanisms have been suggested by which pRB can increase the oxidative capacity of an adipocyte. Augmented Foxc2 expression before induction and early in adipocyte differentiation has been suggested to contribute to

the BAT-phenotype in pRB-deficient MEFs [69] . Further-more, pRB has been reported to bind to the promoter of the gene encoding the PPAR γ -cofactor 1 α (PGC-1 α ) and repress its activity [72] . Another reason for increased UCP1 expression could be the augmented PPAR γ activity in pRB-deficient adipocytes [73] . Through its interaction with C/EBP α , pRB is reported to directly affect the activity of the UCP1 promoter although in a positive manner [74] . Accordingly, pRB can play on many strings when controlling brown adipogenesis and UCP1 expression.

In line with these observations RB1 haploinsufficiency (RB +/- ) in mice also reduced diet-induced obesity and hepatosteatosis. Again this effect was coupled to increased oxidative metabolism most probably caused by transcriptional induction of genes involved in energy metabolism such as PGC-1 α , UCP1, and NRF1 (nuclear respiratory factor 1) (M Louisa Bonet and Andreu Palou, pers. commun.). Interestingly, these data provide evidence that partial pRB deficiency attenuates the development of HFD-induced obesity and obesity-associated metabolic disturbance. Thus, RB haploinsufficiency protected mice against HFD-induced insulin resistance. Moreover, RB +/- mice fed regular chow also displayed signs of increased insulin sensitivity when

Mesenchymalstem cell

PPARγ

PGC-1α/βPGC-1α/β

C/EBPα

PRDM16

Myoblast

RIP140

Myocyte

Brownadipocyte

Brown-likeadipocyte

Brownpreadipocyte

Whiteadipocyte

Whitepreadipocyte

PRDM16

PPARγ

Figure 3 . Pathways controlling white versus brown adipogenesis. BMP: Bone-morphogenetic protein; C/EBP: CCAAT/enhancer binding protein; Foxc2: Forkhead box protein c2; Myf5: Myogenic factor 5; MyoD: Myogenic differentiation; PGC-1 α : PPAR γ -cofactor 1 α ; PPAR γ : Peroxisome proliferator-activated receptor γ ; pRB: Retinoblastoma protein; PRDM16: Positive-regulatory-domain-containing 16; RIP140: Receptor interacting protein 140.

compared with wild-type control animals. Under these conditions increased insulin sensitivity was not caused by differences in body adiposity, and since metabolism is increased, whole-body energy expenditure may be connected to pancreatic beta cell function (M Louisa Bonet and Andreu Palou, pers. commun.). Another possibility is that pRB affect insulin signaling more directly since levels of activated protein kinase B are increased in pRB-deficient MEFs [75] . In addition, lack of pRB may affect insulin sensitivity via PPAR γ since PPAR γ and C/EBP α cooperate to increase insulin sensitivity [76] . Thus, some of the effects of pRB on adipocyte function can be explained by changes in PPAR γ activity. Moreover, pRB has been shown to interact directly with C/EBPs in adipocytes [30] thereby opening another possible mechanism by which pRB could affect insulin sensitivity. Thus, pRB can regulate insulin sensitivity through a number of interaction partners.

In summary, pRB is involved in regulation of the oxidative capacity of the adipocyte, both early, through controlling white versus brown adipogenesis, and later in the mature adipocyte. Furthermore, recent data suggests that pRB play a role in the regulation of adipocyte insulin sensitivity.

3.2 p53 in adipocyte function Although p53 stand as one of the best described genes, very little is known about its role in adipocytes. Only a few reports have indicated that this transcription factor might have a role to play in adipocyte biology. Two N-terminal residues in p53 which normally are phosphorylated upon activation of p53 were found to be phosphorylated late in adipogenesis [77] . Furthermore, both the mRNA and protein levels have been shown to be upregulated in the adipose tissue of obese mice [78] . Lastly, mice deficient for p53 have increased expression of sirtuin 1 (SIRT1) in their adipose tissue [79] . These observations all indicate that p53 is important for proper adipocyte biology. Such a function of p53 would have been unheard of some years ago when p53 was regarded as a dormant guardian only awakened upon severe cellular stress. Today, several reports have however shown an involvement of p53 in regulating metabolism [80] .

In 1931, Otto Warburg discovered that cancer cells preferentially used the glycolytic pathways for energy generation [81] . Absence of p53 activity has been suggested to account for the Warburg effect as liver mitochondria from p53-deficient mice had decreased oxygen consumption. This finding was recapitulated along with the demonstration of increased glycolysis in p53-deficient colon cancer cells [82] . Increased glycolysis was at least in part explained by decreased expression of synthesis of cytochrome c oxidase 2 (SCO2), a gene involved in oxidative phosphorylation, and/or TP53-induced glycolysis and apoptosis regulator (TIGAR), an inhibitor of glycolysis, in p53-deficient cells. Both genes are regulated by p53 [82,83] . Whether p53-deficient adipo-cytes also rely on glucose for ATP generation is an open question. This would, however, fit poorly with the increased

SIRT1 expression in adipose tissue of p53-deficient mice as augmented levels of this enzyme have been shown to cause increased lipolysis in adipose tissue [84] . It is, however, conceivable that p53 exerts opposite functions with respect to metabolic regulation in liver and adipose tissue. For example, SIRT1 is repressed by p53 in the adipose tissue which is not the case in the liver. In contrast, p53 is required for upregulation of SIRT1 expression in the liver during fasting [79] . Further studies are clearly required in order to elucidate the role of p53 in regulating adipocyte biology.

4. Conclusion

A better understanding of the pathways involved in adipo-cyte differentiation and function is a prerequisite for curbing the epidemic of obesity that has been flourishing in recent years. Of special interest is the development of BAT which has the ability to dissipate energy in the form of heat. Recent findings concerning the pathways determining white versus brown adipogenesis are summarized in Figure 3 .

It has within recent years become evident that the two tumor suppressors, pRB and p53, are involved in both the development and function of adipocytes. Interestingly, pRB is not only required for adipose conversion but this pocket protein is also involved in determining the mitochondrial capacity of the adipose tissue. In contrast to pRB, p53 inhibits adipogenesis and although its role in adipocyte function is less clear, results suggest that p53 is involved in maintaining proper function of adipose tissue.

5. Expert opinion

The explosion in the prevalence of obesity in recent years has spurred great interest into understanding the development and function of the adipose tissue hoping that increased knowledge would facilitate the development of proper treatment. In many ways the development of obesity is simple. It is merely caused by surplus energy intake unmatched by energy expenditure. The simplicity does, however, not reflect the process of adipogenesis. Several genes are reported to participate in the development and function of adipose tissues. Here we have discussed the involvement of the two famous tumor suppressors, pRB and p53, in adipocyte biology.

Due to their essential function in upholding cell cycle control, direct targeting of pRB and p53 for treatment of obesity obviously seems a very unwise and dangerous strategy. Rather, understanding of their modes of action could lead to the discovery of and/or provide useful information on novel drug targets.

With respect to pRB, its involvement in brown adipo-genesis is of major interest. Delineation of the pathways controlling white versus brown adipogenesis has been of major interest the recent years as the prospects of turning WAT into BAT are promising with respect to treating

obesity. Whereas intrascapular brown adipocyte depots persist into adulthood in rodents, this depot is replaced by white adipocytes in humans and other larger mammals shortly after birth [25] . Thus, it has been generally assumed that healthy adults lack BAT. This view has however been challenged the use of fluorodeoxyglucose positron emission tomography (FDG PET) to trace tumor metastasis [85] . Multiple reports have suggested that uptake of FDG in the neck and upper chest region may be in brown fat [86-88] . Pre-treatment with a single dose of propranolol blocks the FDG uptake in brown adipose tissue [89] . Moreover, the finding that patients placed at high temperature prior to dosing and during the uptake phase exhibited a significantly reduced FDG uptake in the areas presumed to represent BAT [90] strongly suggests that the brown adipocytes also in humans are temperature sensitive. Thus, increasing expression of UCP1 and genes associated with a brown-like adipocyte phenotype may protect against obesity in humans also. It is also worth noting that UCP1 mRNA has been detected in all intra- and extraperitoneal adipose tissues in adult humans, and it has been estimated that 1 in 100 – 200 adipocytes in human intraperitoneal adipose tissue expresses UCP1 [64] , and several reports indicate that brown-like multilocular adipocytes are interspersed within the white adipose depots in adult humans [62-64] . Interestingly morbidly obese subjects have a significantly lower UCP1 mRNA expression level in adipose tissue [64] and a single nucleotide polymorphism in the human UCP1 gene has been shown to be associated with propensity to weight gain [91] . In addition, gene expression analysis has suggested that a reduced brown adipose phenotype and lower expression of Foxc2 and, paradoxically, pRB is also associated with insulin resistance in humans [92] .

Regarding p53 and adipocyte biology, its target gene SIRT1 shows the most interesting characteristics regarding the treatment of obesity and associated diseases. SIRT1 is

member of a family of NAD + -dependent deacetylases. It has been a subject of major interest in recent years due to its positive effect on longevity and insulin sensitivity [93] . Resveratrol, which activates the deacetylase, has beneficial effects on survival and metabolic disorders in mice fed a high-calorie diet [94,95] . The search for more potent activators than resveratrol is currently ongoing and results with respect to their effect on insulin sensitivity and mitochondrial capacity are promising [96] .

Turning the picture upside down, the knowledge from the studies on pRB and p53 in adipocyte biology could be useful in cancer treatment. The increased adipogenic potential of fibroblasts lacking p53 could prove useful when treating cancers of a fibroblastic origin. Treating tumors with deletions in p53 with an adipogenic agent could theoretically lead to at least a partial differentiation of tumor cells, giving them a less malignant phenotype. The PPAR γ ligand rosiglitazone has already proven useful in the treatment of patients with breast carcinoma [97] . The same would be the case with ablation of pRB. Based on the observation by Auwerx and colleagues these cells would have enhanced PPAR γ activity [73] and would presumably respond better to rosiglitazone treatment compared with tumors with a functional pRB.

Acknowledgments

Research in the authors’ laboratory has been supported by The Danish Natural Science Research Council, The Novo Nordisk Foundation and The Danish Council for Strategic Research.

Declaration of interest

The authors declare no conflict of interest and have received no payment in preparation of this manuscript.

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•• The fi rst study, to our knowledge, showing that myocytes and brown adipocyte originate from the same lineage.

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•• This elegant study illustrates the importance of PRDM16 in controlling brown adipocyte determination and differentiation.

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• An excellent review describing recent advances in the understanding of adipocyte differentiation.

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• A comprehensive review summarizing knowledge on white versus brown adipogenesis.

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•• An important study showing the importance of BMP7 in the determination and differentiation of brown adipocytes.

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• A study illustrating the linkage between pRB and C/EBPs during adipose conversion.

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• An elegant study reporting on a surprising role for XOR in the generation of PPARg ligand(s) during adipogenesis.

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•• A study illustrating both stimulatory and inhibitory effect of the respective E2F family members on adipose conversion.

37. Porse BT, Pedersen TA, Xu X, et al. E2F repression by C/EBP α is required for adipogenesis and granulopoiesis in vivo. Cell 2001 ; 107 : 247 -58

• A study showing that the ability of C/EBPa to repress E2Fs is important for its adipogenic potential.

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• This study shows that the white adipocytes in mice with adipose-specifi c expression of Foxc2 have increased mitochondrial capacity.

56. Tsukiyama-Kohara K, Poulin F, Kohara M, et al. Adipose tissue reduction in mice

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• A study showing that knockout of 4E-BP1 results in white adipocytes with a brown-adipocyte-like phenotype.

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• A study reporting on the cloning of the brown adipose marker Cidea and its importance in regulating energy dissipation through UCP1 inhibition.

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•• An in vivo study showing the importance of pRB in controlling the mitochondrial function in adipocytes and energy expenditure.

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• The fi rst report, to our knowledge, on the involvement of pRB in regulating white versus brown adipogenesis.

70. Seale P, Kajimura S, Yang W, et al. Transcriptional control of brown fat determination by PRDM16. Cell Metab 2007 ; 6 : 38 -54

•• The fi rst study, to our knowledge, showing the importance of PRDM16 in controlling brown adipose conversion.

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• A study showing the involvement of the pRB family member p107 in controlling brown adipogenesis.

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• This study shows repression of PPARg activity by pRB through a direct interaction.

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78. Yahagi N, Shimano H, Matsuzaka T, et al. p53 activation in adipocytes of obese mice. J Biol Chem 2003 ; 278 : 25395 -400

• The fi rst study, to our knowledge, linking p53 to a possible role in controlling adipocyte function.

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• A study linking p53 to the Warburg effect.

83. Bensaad K, Tsuruta A, Selak MA, et al. TIGAR, a p53-inducible regulator of glycolysis and apoptosis. Cell 2006 ; 126 : 107 -20

84. Picard F, Kurtev M, Chung N, et al. Sirt1 promotes fat mobilization in white adipocytes by repressing PPAR- γ . Nature 2004 ; 429 : 771 -6

• The fi rst study, to our knowledge linking SIRT1 to adipocyte function.

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• A review summarizing evidence for the presence of BAT in adult humans.

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• A study reporting on the benefi cial effect of the SIRT1-activator resveratrol on several metabolic parameters.

95. Lagouge M, Argmann C, Gerhart-Hines Z, et al. Resveratrol improves mitochondrial function and protects against metabolic disease by activating SIRT1 and PGC-1 α . Cell 2006 ; 127 : 1109 -22

• A study reporting on the benefi cial effect of the SIRT1-activator resveratrol on several metabolic parameters.

96. Milne JC, Lambert PD, Schenk S, et al. Small molecule activators of SIRT1 as therapeutics for the treatment of type 2 diabetes. Nature 2007 ; 450 : 712 -6

• A study reporting on the benefi cial effect of synthetic SIRT1-activators on several metabolic parameters.

97. Mueller E, Sarraf P, Tontonoz P, et al. Terminal differentiation of human breast cancer through PPAR γ . Mol Cell 1998 ; 1 : 465 -70

• A study showing the ability of a potent PPARg ligand to force cancer cells into a less malignant state.

98. Madsen L, Petersen RK, Sorensen MB, et al. Adipocyte differentiation of 3T3-L1 preadipocytes is dependent on lipoxygenase activity during the initial stages of the differentiation process. Biochem J 2003 ; 375 : 539 -49 .

Affi liation Philip Hallenborg 1 PhD , Søren Feddersen 1 PhD , Lise Madsen 1, 2 PhD & Karsten Kristiansen † 1, 3 MD, Professor † Author for correspondence 1 University of Southern Denmark, Department of Biochemistry and Molecular Biology, Campusvej 55, 5230 Odense M, Denmark 2 National Institute of Nutrition and Seafood Research, PO Box 2029 Nordnes, 5817 Bergen, Norway †3 University of Copenhagen, Department of Biology, Ole Maaløes Vej 5, 2200 Copenhagen N, Denmark Tel: +45 3532 4443 ; Fax: +45 6550 2467 ; E-mail: kk@bio.ku.dk

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