International Journal of Obesity (1999) 23, 1±6 ß 1999 Stockton Press All rights reserved 0307±0565/99 $12.00 http://www.stockton-press.co.uk/ijo Retinoids and adipose tissues: metabolism, cell differentiation and gene expression

F Villarroya*1, M Giralt1 and R Iglesias1

1Departament de BioquõÂmica i Biologia Molecular, Universitat de Barcelona, Barcelona, Spain

Vitamin A derivatives (retinoids) play a key role in mammalian development and cell differentiation. Isomers of retinoic acid, the main active metabolite of vitamin A, activate retinoid receptors that act as ligand-dependent transcription factors and affect gene expression. White and brown adipose tissues are major sites of storage of vitamin A derivatives and they play an active role in whole body metabolism of retinoids. Moreover, adipose tissues are targets for the action of retinoic acid. In most cases retinoic acid impairs adipocyte differentiation although its ®nal effects on adipose tissue development depend on retinoic acid concentration, isomers availability and expression of retinoid receptor subtypes in the white or brown adipocyte. Retinoic acid (RA) has a speci®c effect on brown adipose tissue, because it activates transcription of the gene for uncoupling protein-1, responsible for brown fat thermogenesis.

Keywords: vitamin A; retinoic; adipose tissue; uncoupling protein

Introduction homodimer or as a heterodimer of RXR and some members of the nuclear receptor superfamily, for example, PPARs. In contrast, when RXR hetero- Vitamin A and its structural derivatives (retinoids) dimerizes with VDR and T3R, the action of 9-cis- exert profound effects on vision, embryonic develop- RA on the RXR moiety is suppressed. With respect to ment, reproduction, haematopoiesis, and the regula- RAR=RXR heterodimers, the ligand-induced tran- tion and differentiation of mammalian epithelial and scriptional activation for RXR remains controversial. mesenchymal cells. Other than in vision, most of these Besides the existence of two isomers of RA, whose effects occur through retinoic acid (RA), the main synthesis might be differentially regulated, additional active metabolite derived from vitamin A by enzy- levels of complexity in the RA signalling pathway matic oxidation of retinol. Two isomers of RA, all- must be considered. Both the RAR and the RXR trans RA and 9-cis RA, are biologically active and subfamilies are composed of three different subtypes, most of the cells studied to date can convert either of a, b and g, which are encoded by different genes and the RA isomers into the other. The biological effects show characteristic developmental- and tissue-speci®c of RA isomers involve the activation of ligand-depen- regulated expression (for reviews see Refs 1,2). dent transcription factors, the retinoid receptors. Furthermore, a cross-talk between the RA signalling These belong to the nuclear receptor superfamily, pathway and signalling pathways mediated by cell- 3 which includes the steroid, vitamin D (VDR) and T3 surface receptors has been postulated. A diagram of receptors (T3R), the peroxisome proliferator-activated the major mechanisms of action for RA on gene receptors (PPARs) and a number of orphan receptors expression is shown in Figure 1. whose ligands are unknown. Indeed, two different subfamilies of nuclear receptors, the retinoic acid receptors (RARs) and the retinoid-X receptors (RXRs) mediate the actions of RA isomers. All- trans-RA in¯uences gene expression by activating Retinoids in adipose tissues the RAR moiety of RAR=RXR heterodimers that bind RA response elements. 9-cis-RA is considered There are two kinds of adipose tissue in mammals, as a `pan-agonist': it binds RAR and RXR, and with different physiological roles. White adipose in¯uences gene expression by activating RAR (in tissue (WAT) stores energy in the form of triacy- the same way as all-trans-RA), but also by activating lglycerols. Brown adipose tissue (BAT) in contrast, is RXR when it interacts with target genes as an RXR the main site of non-shivering thermogenesis. This function of BAT is due to uncoupling protein-1, a natural uncoupler of the oxidative phosphorylation *Correspondence: Francesc Villarroya, Departament de system present exclusively in BAT mitochondria. At BioquõÂmica i Biologia Molecular, Universitat de Barcelona, Diagonal 645, 08028-Barcelona, Spain. least in rodents, high activity of BAT correlates Received 28 May 1998; revised=accepted 28 October 1998 negatively with the development of obesity, that is, Retinoids and adiopose tissues F Villarroya et al 2 and white adipocytes synthesize RBP in vitro and secrete it into the culture medium7 and retinol can also be mobilizated from adipocytes.12 Thus, taken together, these data suggest that adipose tissues reg- ulate retinoid homeostasis, as they are involved not only in retinol uptake and storage, but also in its mobilization and transport. As stated in the introduction, most of the effects of retinoids are mediated by its active form, RA. Differ- ent adipose depots contain RA at concentrations similar to those determined in liver and in other RA-responsive tissues.13 Thus, considering the total Figure 1 Schematic representation of retinoids metabolism in mass of adipose tissues, they may be quantitatively the adipocyte and the mechanisms of action of all-trans retinoic important for RA metabolism. In relation to the origin acid (RA) and 9-cis RA on gene transcription. CRBP ˆ cellular of the RA present in adipose tissues, it has been retinol-binding protein; PPAR ˆ peroxisome proliferator-acti- vated receptor; RAR ˆ RA receptor; RBP ˆ retinol-binding pro- estimated that RA is derived mainly from local tein; RXR ˆ retinoid-X receptor. synthesis.13 It is considered that retinol is enzymati- cally oxidized to RA by mechanisms similar to those acting in other retinoid-sensitive tissues.14,15 In addi- tion, systemic RA, which circulates in plasma bound an increase in WAT mass (for a recent review see Ref. to albumin, also contributes to the RA present in the 4). In addition, both WAT and BAT produce hormo- adipose tissue.13 Figure 1 depicts the pathways of nal signals. WAT secretes leptin and tumor necrosis generation for active forms of retinoids. factor (TNF), that constitute regulatory signals for Adipose tissues have long been recognized as a energy homeostasis and most probably for other potential site for RA action, and retinoid receptors physiological functions.5 BAT is an extra-thyroidal belonging to the two different subfamilies, RAR and 16,17 source of T3, the biologically active form derived RXR, are expressed in adipose tissues. However, from the main product of the thyroid gland, T4. WAT and BAT show a differential pattern of expres- BAT T3 production is modulated in parallel with the sion for RAR and RXR subtypes: at the mRNA level, thermogenic status of the tissue and can be viewed as RARa, RARg and RXRa are highly expressed in a signal to increase basal metabolic status in other WAT depots, whereas RARb and RXRg are poorly tissues.6 Moreover, recent studies have indicated that expressed. In contrast, both RARb and RXRg mRNAs both adipose tissues, WAT and BAT, may play a key are abundant in BAT, while RARa, RARg and RXRa role in retinoid uptake, storage, mobilization and are expressed at lower levels in BAT than in WAT.18 transport. The characteristic pro®le of expression of RAR and The liver is the main organ involved in the storage RXR subtypes in BAT vs WAT is the same in several and homeostasis of retinoids; however, adipose tissues mammalian species, from rodents to the baboon.18 contain substantial amounts of retinol and retinyl Thus, the conservation of the expression pattern of esters, and it has been estimated that all adipose retinoid receptors in WAT vs BAT suggests a speci®c depots could account for 15 ± 20% of the total body function for the various retinoid receptor subtypes in retinoid stores.7 Retinoids accumulate in adipocytes the control of gene expression in white and brown rather than in stromal-vascular cells7 and differentia- adipocytes. Although the speci®c role of the different tion of precursor cells into white or brown adipocytes receptors subtypes in WAT with respect to BAT has is associated with accumulation of intracellular reti- not been studied, it has been described that the noids.8 Adipose tissue retinoid stores may be derived targeted disruption of particular RXR and RAR sub- from two different sources. First, retinol can be types, either alone or in combination, causes complex provided by the lipoprotein lipase hydrolysis of the effects in the development of other tissues and in retinyl esters contained in postprandial chylomicrons.9 morphogenesis.19 It is considered that this pathway is especially impor- tant in both WAT and BAT. Second, retinol can be taken up by cells from the bloodstream where it is transported by retinol-binding protein (RBP). RBP Retinoic acid and adipocyte forms a protein complex with another plasma protein, differentiation transthyretin.10 Holo-RBP delivers retinol to target cells via the cellular retinol-binding protein (CRBP), also believed to be crucial in retinol metabolism, as In 1980, RA was ®rst recognized as a potent inhibitor the retinol-CRBP complex is considered the optimal of adipocyte differentiation.20 When pre-adipose cells substrate for either retinol esteri®cation or oxidation from different cell lines such as 3T3-L1 or 3T3- to RA.11 Interestingly, CRBP and RBP are highly F442A are exposed to all-trans RA at concentrations expressed in both WAT and BAT.7 Moreover, brown ranging from 3 nM to 10 mM, the appearance of the Retinoids and adiopose tissues F Villarroya et al 3 characteristic features of the white adipose cell pheno- might contribute to impaired adipogenesis. Thus, type is blunted.20 ± 22 Lipid accumulation is suppressed RA causes a sustained rise in the expression of c- and molecular markers of adipocyte differentiation jun in adipocyte precursor cells, a transcription such as adipocyte lipid-binding protein or stearoyl- factor with anti-adipogenic properties.27 RA also CoA desaturase mRNAs22 are down-regulated. The causes a down-regulation of thyroid hormone recep- effects of RA on the adipose cells have however two tors in association with inhibition of Ob17 adipo- particular features. First, addition of RA to cell genic differentiation28 and this might contribute to cultures is only effective in blocking cell differentia- inhibition of adipocyte differentiation considering tion when performed at early stages of the pro- the positive action of thyroid hormones on adipo- cess.22,23 Second, the analysis of marker genes, genesis. Moreover, RA has been reported to down- whose expression is characteristic of the differentiated regulate the expression of molecular markers of phenotype of adipocytes, does not provide evidence of adipogenesis such as adipsin29 or lipoprotein direct effects of RA on gene transcription through lipase,30 through post-transcriptional mechanisms potential `negative RA responsive elements'. that await characterization. The differentiation program of the white adipo- The physiological role of the inhibition by RA of cyte, has been extensively studied in cell cultures adipocyte differentiation has been questioned by and several transcription factors have been identi®ed observations that the effect of low doses (1 ± 10 nM) which act sequentially and cooperatively to trigger of all-trans RA on adipogenesis changes from inhibi- adipocyte-speci®c gene expression and elicit the tory to enhancing.31,32 It has been argued that these acquisition of the adipocyte phenotype. Basically, concentrations are in the range of estimates of RA the induction of adipocyte differentiation involves concentration in adipose tissues13 and of the Kd the interplay of members of the CAAT=enhancer values of RARs for all-trans RA33 and therefore binding protein (C=EBP) and PPAR families of they are more likely to be related to the concentrations transcription factors. C=EBP proteins are members of the retinoic acid to which the cell nuclei are of the b-ZIP family of transcription factors, whereas exposed during development and differentiation. PPARs are a subgroup of nuclear hormone recep- However, the molecular mechanisms by which low tors. Several lines of evidence indicate that adipo- concentrations of RA stimulate adipogenesis, have not cyte differentiation involves induction of the been determined. On the other hand, recent ®ndings transcription factor C=EBPb followed by induction point out that retinoids selectively activating RXR of PPARg and ®nally C=EBPa. A complex interplay receptors promote adipogenesis.34 The PPAR=RXR of regulation for expression of the three correspond- complex is dependent on ligand activation not only of ing genes, is considered to lead to the synergistic PPARg but also of RXR.35 9-cis RA can activate both action of PPARg and C=EBPa that activates adipo- RAR and RXR receptors and all-trans RA is able to cyte-speci®c genes. The whole process has recently isomerize to 9-cis RA in most cells studied so far.36 It been reviewed.24 How RA interferes with this pro- appears that the resulting effects of retinoids on cess and blocks adipogenesis is not fully under- adipose differentiation depend on a complex balance stood. RA is unable to block adipogenesis if between RA metabolism and relative RAR and RXR C=EBPa has already been induced.25 On the other receptor availability in the cell. In fact, expression of hand, RA does not affect the early induction of RXR isoforms is up-regulated during adipocyte dif- C=EBPb, but it blocks the induction of C=EBPa and ferentiation.37 In addition, reduced RAR expression it is a potent inhibitor of PPARg expression.25 Thus, may allow RXR to form heterodimers with PPARg it is proposed that RA blocks C=EBPb-dependent instead of RXR=RAR dimers. activation of expression of PPARg and C=EBPa, which leads to inhibition of adipose differentiation. The lack of sensitivity of pre-adipose cells to inhibition of adipogenesis, in the late stages of differentiation, is explained by the fact that such a mechanism does not operate when the expression of C=EBPa and PPARg has been induced. This propo- sal is schematized in Figure 2. The mechanism for this effect of RA remains unknown. It has been established that activation of RAR receptors, espe- cially the RARa isoform,26 is required for this process, but binding of RAR to DNA target sequences does not appear to be required.25 On the other hand, the down-regulation of RARa and RARg expression that occurs during adipocyte dif- Figure 2 Model illustrating the cascade of transcription factors ferentiation,17 appears to contribute to this desensi- involved in adipocyte differentiation and the sites of action of all- trans retinoic acid (RA) and 9-cis RA. C=EBP ˆ CAAT=enhancer tation to RA of differentiated adipose cells. RA binding protein; RAR ˆ RA receptor; RXR ˆ retinoid-X receptor; action also affects other signalling pathways that PPAR ˆ peroxisome proliferator-activated receptor. Retinoids and adiopose tissues F Villarroya et al 4 Retinoids and brown adipose tissue: central region contains binding sites for RAR and RXR and has been described to mediate responsive- Retinoic acid activates the ness to all-trans RA and 9-cis RA. However, these expression of the uncoupling sites do not ®t with either RAR- or RXR-dependent protein-1 gene canonical DNA sequences for RA-response elements and their precise architecture has not been estab- lished.39,40 A third downstream region binds thyroid The action of RA on brown adipocytes has speci®c hormone receptor, as well as RXR, even though there features, since it promotes the expression of the gene is no evidence of its involvement in mediating retinoid for uncoupling protein-1 (ucp-1), the mitochondrial stimulation through RXR activation.38,43 Figure 3 protein that confers its speci®c thermogenic function summarizes the present knowledge on the mechan- to BAT.38 The potency of RA inducing ucp-1 expres- isms of action of RA on the transcription of the ucp-1 sion, is as high as that of noradrenaline, the classical gene, based on studies of the corresponding rat and agent stimulating ucp-1 gene expression in response mouse genes. to sympathetic activity and thermogenic require- ments.38 The action of RA on the ucp-1 gene occurs through pathways distinct from adrenergic regulation and does not require protein synthesis.38 The expres- Conclusion sion of ucp-1 gene is controlled by two regions upstream from the transcription initiation site: a prox- imal regulatory region and a distal enhancer, which Even though the action of RA promoting ucp-1 expression has been observed in mice treated with are required for hormonal and differentiation-depen- 44 dent regulation (see Figure 3). RA stimulates ucp-1 pharmacological doses of retinoids, the physiologi- gene transcription through a `retinoid responsive cal signi®cance of this effect is unknown. BAT is a region' in the distal enhancer.38 ± 40 site for diet-induced thermogenesis and further The mechanisms of action of RA on the ucp-1 gene, research is needed to determine the effect of vitamin involve a complex array of RA isomers, retinoid A-related nutrients on this function. In fact, carote- noids have been reported to mimic the action of RA receptors and gene elements. Activation of both 45 RAR- and RXR-dependent pathways contributes to on ucp-1 expression in brown adipocytes. Further- the induction of the ucp-1 gene by retinoids, as shown more, the molecular mechanisms for the initiation of by the action of selective agonists of both types of ucp-1 gene expression during ontogeny, which occurs receptor and the ability of overexpressed receptors to before birth in most species, have not been established enhance responsiveness to retinoids.38,41 Moreover, and appear to be independent of adrenergic stimula- each subtype of the receptors RAR or RXR has a tion. RA is a potent morphogen in mammals and its different capacity to mediate the action of retinoids on potential involvement in the ®rst induction of ucp-1 the ucp-1 gene.41 At least three sites inside the gene transcription deserves future research. `retinoid responsive region' may contribute to the RA effects. An upstream region contains a binding Acknowledgements site for PPAR=RXR heterodimers, potentially sensi-  42 Supported by DGICYT, Ministerio de Educacion y tive to 9-cis RA activation of the RXR moiety. A Cultura, Spain (Grant PB95.0960) and Generalitat de Catalunya (Grant 97SGR-187).

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