~ ) Pergamon Pharmac. Ther. Vol. 61, pp. 399--411, 1994 Copyright © 1994 Elsevier Science Ltd

Printed in Great Britain. All rights reserved 0163-7258/94 $26.00

Associate Editor: P. K. CHIANG

MOLECULAR EVENTS IN ADIPOCYTE DEVELOPMENT

S. C. BUTTERWITH Agricultural and Food Research Council, Roslin Institute (Edinburgh), Department of Cellular

and Molecular Biology, Roslin, Midlothian, EH25 9PS, U.K.

Abstraet--Adipocyte hyperplasia occurs by the proliferation and differentiation of adipocyte precursor cells or preadipocytes. Although the process of commitment to the adipocyte lineage is poorly understood, a great deal of information has accumulated about the processes and regulatory mechanisms involved in preadipocyte differentiation. The differentiation of preadipocytes is known to be characterized by increased transcription of a number of specific genes. AP-I and C/EBP binding sites within these genes have been identified as important regulatory sequences. In addition, a specific enhancer sequence has been shown to confer adipose tissue specificity. This article will review the changes in gene transcription that occur during preadipocyte differentiation and how these are regulated. The potential role of autocrine/paracrine acting factors in the proliferation and differentiation of the preadipocyte is also discussed.

Keywords--Growth factors, adipogenesis, differentiation, transcription factors, obesity, pros- taglandins.

CONTENTS

1. Introduction 2. Origin and Development of the Adipocyte 3. Molecular Markers of Differentiation 4. Transcriptional Regulation of Preadipocyte Differentiation 5. The Role of Growth Factors as Autocrine/Paracrine-acting Regulators of

Adipocyte Development 6. The Role of Prostaglandins as Autocrine/Paracrine-acting Regulators of

Adipocyte Development 7. Conclusions and Future Directions Acknowledgements References

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1. I N T R O D U C T I O N

One of the main functions of adipose tissue is to accumulate triacylglycerol to act as an energy store in times of need. However, recent evidence suggests that it also has a number of other important functions. For example, adipose tissue is able to accumulate, store and, when required, mobilize large amounts of unesterified cholesterol (Krause and Hartman, 1984). It also has a

Abbreviations--AE-l, activating element-l; aFGF, acidic fibroblast growth factor; AP-I, activator protein-1; aP2, adipocyte lipid-binding protein; bFGF, basic fibroblast growth factor; CAT, chloramphenicol acetyltransferase; C/EBP, CCAAT/enhancer-binding protein; EGF, epidermal growth factor; FGF, fibroblast growth factor; FSE, fat-specific element; G-3-PDH, glyceroi-3-phosphate dehydrogenase; IGF, insulin-like growth factor; PDGF, platelet-derived growth factor; PGE2 prostaglandin E2; PGF~, prostaglandin F2~; PLA2, phospholipase As; TGF, transforming growth factor; TNF, turnout necrosis factor.

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number of links with the immune system. A number of components of the alternative complement pathway are synthesized in adipose tissue. These include complement factors D (adipsin), C3, and B (Bortell et al., 1992). The presence of tumour necrosis factor (TNF)-0t, an immune regulator, has also been described in adipose tissue; its expression is increased in a number of models of genetic obesity (Hotamisligil et al., 1993). The interaction of adipose tissue with the immune system is likely to be a major area of interest over the coming years.

A large research effort, over a number of years, has been aimed at trying to elucidate the mechanisms involved in adipose tissue development. This work has been carried out using a number of different species of experimental animal, the major aims being: (i) to identify new strategies for the treatment of obesity and other disorders in which overproduction of adipose tissue is a contributory factor and (ii) to develop methods for manipulating the carcass fat content of farm animals. Interest in the regulation of adipocyte development is likely to increase as other functions besides triglyceride storage are described.

This review will describe the molecular events that occur during the development of the adipocyte and how these are regulated at the transcriptional level. Specifically, this will deal with white adipocyte development. Much less is known about brown fat development, and the reader is referred to Ailhaud et al. (1992) for a review. The role of circulating hormones (e.g. growth hormone, insulin etc.) and steroids in the regulation of adipocyte development has been reviewed elsewhere (Ailhaud et al., 1992). This review, therefore, will focus more on the potential role of autocrine/paracrine-acting factors, such as growth factors and prostaglandins, and how these are involved in the regulation of the molecular events that contribute to adipocyte development.

2. ORIGIN AND DEVELOPMENT OF THE ADIPOCYTE

Adipose tissue occurs as both discrete depots and within other tissues (e.g. muscle). These depots differ in both their size and time of development. The adipocytes that make up all these depots have a characteristic round, unilocular appearance in which the cytoplasm and nucleus are displaced to the cell periphery by the presence of a large amount of lipid.

From where and how do adipocytes develop? Early experiments involving the administration of [3H]thymidine to rats demonstrated that adipocytes developed by the proliferation and differen- tiation of cells present in the stromal vascular fraction of adipose tissue and that the mature, fully differentiated adipocyte had no capacity for cell division in vivo (Van, 1985). Subsequently, it became possible to isolate these adipocyte precursor cells and culture them in vitro. These cells are able to proliferate and subsequently differentiate into mature adipocytes (Van, 1985). Mature adipocytes, when cultured in vitro, have been shown to have some limited capacity for cell division (Sugihara et al., 1987), but it is likely that most of the hyperplastic capacity of adipose tissue in vivo resides in a population of adipocyte precursor cells.

Considerable data on the development of the adipocyte has come from in vitro cell culture studies. These studies have used either: (i) primary cultures of adipocyte precursor cells, which are prepared by collagenase digestion of adipose tissue and have a limited life span, or (ii) preadipocyte cell lines, which have been isolated and cloned from a number of sources and which can be passaged indefinitely. Most cell lines developed so far are from rodents, and these have been postulated to represent a slightly earlier stage in adipocyte development than adipocyte precursor cells (Ailhaud et al., 1992). The evidence for this is that freshly isolated adipocyte precursors express early markers of differentiation, such as insulin-like growth factor (IGF)I (Doglio et al., 1987), A2COL6/pOb24 (Dani et al., 1989) and lipoprotein lipase, whereas these markers are absent in preadipocyte cell lines prior to confluence. It has been assumed that primary preparations of adipocyte precursors represent a mixture of different stages of differentiation, although detailed studies using, for example, in situ hybridization have not been performed to confirm this. There are advantages and disadvantages to using either cell lines or primary cultures of adipocyte precursor cells. The major advantage of cell lines is that they are clonally derived and so are a defined homogeneous population at the same stage of differentiation. In contrast, some preparations of adipocyte precursor cells are heterogeneous populations containing other cell types. However, an important

Molecular events in adipocyte development 401

advantage of primary cells is that they may more closely resemble those preadipocytes that are present in vivo.

Preadipocyte cell lines are committed to the adipocyte lineage, and there is no evidence that they can give rise to other cell types. Recent experiments indicate that adipocyte precursor cells may not be irreversibly committed to the adipocyte cell lineage. Culture of endoderm on cells of the stromal vascular fraction prepared from white or brown adipose tissue induces the differentiation of endoderm, which then induces differentiation of stromal-vascular cells into lamina propria cells and chondrocytes (Loncar, 1992). It is not clear from these experiments which cells of the stromal-vascular fraction are differentiating into lamina propria cells and chondrocytes. The preparation of stromal-vascular cells may contain pluripotent stem cells. However, this fraction does contain a high proportion of adipocyte precursor cells, and it would seem likely that some, or all, of these give rise to the different cell types. Further studies are required to answer these questions.

Very little is known about the processes that commit a cell to the adipocyte lineage. Preadipocytes are probably derived from a pluripotent stem cell, the nature and origin of which have yet to be identified. In vitro evidence to support this derives from treatment of 10T1/2 and 3T3 cells with 5-azacytidine, a DNA methylation inhibitor, or 3T3-L 1 cells with 3-deazaadenosine (Chiang, 1981). Subsequently, these cells were able to differentiate into chondrocytes, adipocytes or myoblasts (Sager and Kovac, 1982). Transfection of high molecular weight DNA prepared from preadipocytes or adipose tissue into cells that have no adipogenic potential induces these cells to form adipocytes (Chen et al., 1989). Two genomic clones of 1.2 and 0.6 kb, which confer adipogenic potential, have now been isolated and sequenced, but, as yet, no mRNA or protein has been identified (Colon-Teicher et al., 1993).

3. MOLECULAR MARKERS OF DIFFERENTIATION

Preadipocyte or adipocyte precursor differentiation is characterized by an increased lipogenic capacity and a change from a fibroblast morphology to the unilocular appearance of the mature adipocyte. In vitro, this occurs after cells have reached confluence, and is triggered not by cell contact, but by growth arrest at the Gl/S stage of the cell cycle (Ailhaud et al., 1989). The differentiation process is characterized by the induction and increased expression of a number of specific mRNAs as well as a large accumulation of lipid. Not all differentiation markers appear upon growth arrest; some are switched on and off at specific times.

The earliest differentiation marker so far identified is pOb24. The pOb24 cDNA was isolated from the Ob 1771 cell line by differential screening between early differentiated and undifferentiated exponentially growing cells (Dani et al., 1989). Expression of pOb24 increases rapidly during the early stages of differentiation, and then decreases at a time when late markers of differentiation, such as glycerol-3-phosphate dehydrogenase (G-3-PDH) and adipsin, are increasing. The original pOb24 cDNA clone that was isolated detected a 6 kb mRNA species (Dani et al., 1989), but further studies have isolated a cDNA clone that also detects a 3.7 kb mRNA (Ibrahimi et al., 1993), which has been named A2COL6. Both these mRNA's are derived from the same gene by the use of two different polyadenylation sites.

Expression of A2COL6/pOb24 is not confined to adipose tissue, and occurs in a variety of adult mouse tissues. Expression is low in adult skeletal muscle, but high in embryonic skeletal muscle. In the C2C12 muscle cell line, A2COL6/pOb24 is induced in growth-arrested confluent cells in a similar pattern to that shown by the master regulatory genes MyoD1 and myogenin. This indicates that A2COL6/pOb24 may play an important role in the differentiation of other tissues, as well as adipose tissue.

Other markers that appear early in the differentiation process are clone 5 (Navre and Ringold, 1988), clone 154 (Jiang et al., 1992) and lipoprotein lipase (Dani et al., 1990; Jiang et al., 1992). Clone 5 expression has been linked to the triggering of preadipocyte differentiation because expression of antisense clone 5 in 3T3-L 1 or TA 1 preadipocytes led to the cells failing to accumulate adipocyte-specific mRNAs and to differentiate morphologically (Wenz et al., 1992). The function of clone 154, as yet, is unknown.

402 S.C. BUTTERWITH

In a number of cell lines, at least one postconfluent cell doubling is required before the emergence of late markers, such as G-3-PDH, acetyl coenzyme A carboxylase, fatty acid synthase, phospho- lipase A 2 (PLA:) and adipocyte lipid-binding protein (aP2). Finally, very late markers appear, such as phosphoenolpyruvate carboxykinase and adipsin (Spiegelman et al., 1983).

Adipsin is a serine protease whose cDNA was isolated by differential screening of preconfluent and differentiated 3T3-L1 cells. It has a 1.1 kb mRNA, which codes for a protein of 37-44 kDa (Flier et al., 1987). Sequence analysis has shown that adipsin has homology to complement factor D, and purified adipsin has complement factor D activity (Rosen et al., 1989). Adipsin appears to be expressed only in white and brown adipose tissue and, to a lesser extent, in sciatic nerve (Cook et al., 1987). The expression of adipsin is reduced dramatically in a number of models of acquired and genetic obesity, and a role in regulating adipocyte metabolism and/or systemic energy balance has been proposed (Flier et al., 1987).

4. TRANSCRIPTIONAL REGULATION OF PREADIPOCYTE DIFFERENTIATION

The identification of a number of genes that are specifically activated during the process of preadipocyte differentiation has enabled more detailed studies into their regulation. Two questions are of particular importance: (i) what determines that a gene is switched on specifically during preadipocyte differentiation? and (ii) which gene sequences confer specific expression to adipose tissue?

Comparison of the Y-flanking region of three genes whose transcription is increased dramatically during differentiation, aP2, G-3-PDH and adipsin, has revealed a 13-base region of consensus, which has been named fat-specific element (FSE) 1. An additional 15-base consensus element occurred between G-3-PDH and aP2, and this was named FSE2. When the FSE2 element is sequentially deleted from aP2 promoter/chloramphenicol acetyltransferase (CAT) constructs that are then transfected into preadipocytes, progressively more expression of CAT is seen in the preadipose state. Competition for FSE2-binding factors, using synthetic FSE2 oligo, has similar effects. This evidence suggests that the FSE2 element acts to suppress the activity of the aP2 gene in preadipocytes (Distel et al., 1987).

The patterns of proteins binding to FSE2 seen in gel retardation assays are markedly different between the preadipose and the differentiated state. Complexes on the FSE2 element in the aP2 promoter appear to involve c-fos, as they can be disrupted by using antibodies raised against c-fos (Distel et al., 1987). This is not the case for FSE2 elements present in other adipocyte differentiation specific genes.

The presence of v-jun, a protein closely related to activator protein-1 (AP-1 or c-jun), has also been demonstrated in the protein complex that binds to FSE2 (Rauscher et al., 1988). It is unclear, at present, how c-fos and v-jun interact with FSE2 and with each other within the complex. A function for c-fos has also been reported in the regulation of another differentiation marker, lipoprotein-lipase, by growth hormone (Barcellini-Couget et al., 1993). Growth hormone treatment is also known to activate c-jun expression in 3T3-LI cells (Gurland et al., 1990). The regions of the mouse LPL gene, which have been sequenced so far, do not contain an AP-1 site, but the chicken LPL gene, which has been completely sequenced, does contain an element that is homologous to the core sequence of FSE2 (Cooper et al., 1992).

The aP2 gene promoter contains a second region (activating element-l, AE-I) that is linked to adipocyte differentiation-specific expression. The binding of both AE-1 and AP-I are required for optimal expression of aP2 during differentiation. The AE-1 region binds the transcription factor CCAAT/enhancer-binding protein (C/EBP). Expression of C/EBP alone is not sufficient to promote preadipocyte differentiation but does cause growth arrest (Umek et al., 1991).

Intestine, liver and adipose tissue are major sites of C/EBP expression, and three types of C/EBP (~t, fl and 6) are induced during adipocyte differentiation (Cao et al., 1991). The pattern of expression, however, is different for the three forms (Cao et al., 1991). Both C/EBP fl and 6 are stimulated by a combination of dexamethasone, methylisobutylxanthine and insulin that is commonly used to stimulate differentiation in 3T3-L 1 cells. Expression of C/EBP fl and 6 decreases when dexamethasone and methylisobutylxanthine are removed. In contrast, the expression of

Molecular events in adipocyte development 403

C/EBP ~, is much later and correlates with the appearance of differentiation markers, such as G-3-PDH and aP2. A number of genes, whose expression is activated during preadipocyte differentiation, have been shown to have C/EBP-binding sites in their promoters (Herrera et al., 1989; Danesch et al., 1992; Lin and Lane, 1992).

Further evidence for a role of C/EBP in the coordinated regulation of differentiation-specific genes is provided by experiments using antisense constructs to block the action of C/EBP in vitro. Antisense C/EBP constructs transfected into 3T3-L1 preadipocytes inhibited C/EBP mRNA and protein production, as well as inhibiting several adipocyte differentiation marker mRNAs (Lin and Lane, 1992) and triglyceride accumulation (Samuelsson et al., 1991; Lin and Lane, 1992). In contrast, the lipoprotein-lipase gene appears not to be regulated by C/EBP during differentiation (Samuelsson et al., 1991). Suppression of C/EBP expression by administration of hydrocortisone to differentiating 3T3-F442A preadipocytes failed to have any effect on lipoprotein-lipase expression. The lipoprotein-lipase gene appears to be regulated by a complicated system of positive and negative regulatory sequences, which are located not only in the Y-flanking region, but also in intragenic and 3'-flanking regions, (Enerback et al., 1992).

Analyses using deletion mutants of the aP2 gene promoter have identified the presence of negative regulatory elements (Distel et al., 1987; Yang et al., 1989) that overlap the C/EBP-binding region (Cheneval et al., 1991). The effect of these negative elements is relieved by treatment of confluent preadipocytes with cAMP, a known activator of differentiation. At some stage during the differentiation process, the effect of the negative element is abolished, and cAMP no longer exerts a stimulatory effect on the aP2 promoter (Yang et al., 1989).

It is unlikely that either AP-1 or C/EBP function in determining adipose-specific expression but, they probably have a modulating role in adipocyte differentiation-specific expression. Both aP2 and adipsin genes are specifically expressed in adipose tissue and, therefore, are a logical starting point to look for gene sequences that confer adipocyte specificity. Constructs containing 5' sequences extending to - 1.7 kb from the transcription start site of the aP2 gene linked to a CAT reporter gene fail to confer adipose tissue specificity in transgenic mice. This region of sequence contains both C/EBP- and AP-l-binding sites, which play a role in differentiation-specific gene expression in vitro (Herrera et al., 1989), and it is clear from these experiments that either these factors are not required for adipose specific expression or they also require other complementary factors. Further analysis of the aP2 gene revealed the presence of a 500-bp enhancer at - 5 .4 to -4 .9 kb, which alone conferred adipose tissue specific expression (Ross et al., 1990). Thus neither the AP-1- or C/EBP-binding sites were required for adipose-specific expression.

Present within this 500-bp enhancer is a shorter sequence of 122 bp, which directs differentiation- specific gene expression. Analysis of this 122-bp sequence has identified a number of important cis-act ing elements (Graves et al., 1992). Two of these (ARE2 and ARE4) bind a nuclear factor that is present in a number of other cell types besides those of the adipocyte. However, another nuclear factor, termed ARF6, binds to two other sequences (ARE6 and ARE7) within this 122-bp sequence and is adipose-specific. Mutation within the ARE6 site greatly reduces the activity of the enhancer. Graves et al. (1992) have postulated that ARF6 might represent an adipocyte master regulator similar to that of the MyoD family of muscle master regulators.

The relationship between the enhancer and the C/EBP-, AP-1- and inhibitor-binding sites are not known. Lane and colleagues (Cheneval et al., 1991) have postulated that the negative element may override the effect of the enhancer until differentiation is induced, but, thereafter, cAMP and C/EBP would act to derepress the promoter. Further studies are needed to confirm this.

5. THE ROLE OF GROWTH FACTORS AS AUTOCRINE/PARACRINE-ACTING REGULATORS OF ADIPOCYTE DEVELOPMENT

A number of growth factors can regulate preadipocyte proliferation and differentiation in vitro. IGF has been the most extensively studied, and has been shown to be a mitogen in both preadipocyte cell lines and primary adipocyte precursors (Deslex et al., 1986; Butterwith and Goddard, 1991). IGF-II has a similar potency to IGF-I in chicken adipocyte precursors (Butterwith and Goddard, 1991). However, the chicken type II receptor does not bind IGF-II (Clairmont and

404 S.C. BUTTERWITH

Czech, 1989; Duclos and Goddard, 1990; Yang et al., 1991), and IGF-II appears to elicit its response through the type I receptor. The effect of IGF-II on preadipocyte proliferation has not been tested in a cell system where IGF-II acts through the type II receptor. In contrast to the IGFs, much higher concentrations of insulin are required to stimulate preadipocyte proliferation. Therefore, insulin exerts its effect via the IGF-I receptor rather than the insulin receptor.

It is unclear, at present, whether the action of IGF-I on preadipocyte proliferation and differentiation is via endocrine or autocrine/paracrine mechanisms or both. IGF-I has been shown to be expressed in adipocyte precursor cells (Gaskins et al., 1990; Burt et al., 1992) and preadipocytes (Doglio et al., 1987) in culture. In primary cultures of adipocyte precursors, IGF-I was expressed during both proliferation and differentiation (Doglio et al., 1987; Burt et al., 1992). However, in Ob 1771 preadipocytes, IGF-I was only expressed during differentiation (Doglio et al., 1987). The reason for this difference is not clear, but might reflect differences between primary cells and cell lines or differences in the sensitivity of the detection methods used. In both cell systems, the expression of IGF-I increased during differentiation. Expression of IGF-I during differentiation can be stimulated by growth hormone (Doglio et al., 1987; Gaskins et al., 1990).

The action of the IGFs on adipose tissue is likely to be influenced by the action of the IGF-binding proteins, of which six have now been characterized (Rechler and Brown, 1992). IGF-binding proteins can either enhance or inhibit the action of the IGFs, and this very much depends on the cell type and particular binding protein under study (for a review, see Clemmons, 1992). The production of IGF-binding proteins has been demonstrated in differentiated pig preadipocytes, using a polyethylene glycol precipitation binding assay (Gaskins et al., 1990). The only study, so far, that attempted to characterize the IGF-binding proteins produced by adipocyte precursors was that of Nougues et al. (1993). They identified binding proteins of 40, 29 and 25 kDa, but were unable to identify which cell type was producing them because of the heterogeneity of their cell cultures. It will be important to identify and characterize the biological function of the IGF-binding proteins in adipose tissue because of the importance of the IGFs in regulating proliferation and differentiation of preadipocytes.

The fibroblast growth factors (FGFs) are a family of growth factors that have been shown to regulate the proliferation of a number of cell types in vitro, but the FGFs also have important functions in regulating a number of other biological processes besides cell proliferation (Baird, 1993). There are a number of different members of the FGF family, of which only basic FGF (bFGF) and acidic FGF (aFGF) have been studied in relation to preadipocytes. Both bFGF and aFGF stimulate preadipocyte proliferation (Serrero, 1987; Aoki et al., 1990; Butterwith et al., 1993), but bFGF has a much greater potency than aFGF (Butterwith et al., 1993). However, the potency of aFGF and bFGF is similar in the presence of added heparin, indicating an important role for the extracellular matrix in FGF action (Butterwith et al., 1993).

bFGF has been shown to be expressed by proliferating and differentiating preadipocytes in culture (Burt et al., 1992; Teichert-Kuliszewska et al., 1992) and in adipose tissue in vivo (Burt et al., 1992). The presence of bFGF protein in conditioned medium from proliferating preadipocytes (Teichert-Kuliszewska et al., 1992) is further evidence for a paracrine/autocrine role. Interestingly, preadipocytes prepared from obese humans have a greater expression of bFGF mRNA compared with their lean counterparts (Teichert-Kuliszewska et al., 1992), and have an increased capacity for cell proliferation (Roncari et al., 1986).

The role of the FGFs in the regulation of preadipocyte differentiation is less clear, and differences exist depending on the cell type and species studied. In the TA1 preadipocyte cell line, for example, differentiation is inhibited by concentrations of FGF greater than 1 ng/mL (Navre and Ringold, 1989). Roncari and Le Blanc (1990) have also shown that rat adipocyte precursor differentiation was inhibited by bFGF, but this was not confirmed by the studies of Serrero (1987), who found stimulation of differentiation by bFGF in adipocyte precursors from the same species. Broad and Ham (1983), working with sheep precursors, and Butterwith et al. (1991), using chicken adipocyte precursor cells, failed to demonstrate an inhibition of differentiation by bFGF. In sheep precursors, FGF was a stimulator of differentiation. A possible explanation is that in both the experiments where an inhibition of differentiation occurred, the cells were cultured in serum-containing medium. An interaction between FGF and an unknown serum factor, therefore, might be important for inhibition.

Molecular events in adipocyte development 405

An endocrine role for pituitary-derived FGFs, or related peptides has been proposed by Roncari (Roncari and Le Blanc, 1990). However, much of the available information suggests that neither bFGF or aFGF are secreted proteins. Both cDNAs for aFGF and bFGF have been sequenced and provide no evidence of a signal sequence (Abraham et al., 1986; Jaye et al., 1986). Biosynthetic studies have also shown these growth factors to be cell-associated (Klagsbrun, 1989). Other pituitary-derived peptides have been described that stimulate preadipocyte proliferation (Lau et al., 1983) and may have an endocrine role, but these peptides have not yet been fully characterized.

Transforming growth factor (TGF)-fl is a potent inhibitor of preadipocyte differentiation in a number of cell lines and primary cells (Ignotz and Massague, 1985; Sparks and Scott, 1986; Gimble et al., 1989; Torti et al., 1989; Dani et al., 1990; Butterwith and Gilroy, 1991). When added to adipocyte precursor cells in vitro, TGF-fl stimulates proliferation (Butterwith and Goddard, 1991; Richardson et al., 1992). It is also able to synergize with a number of other growth factors (Butterwith and Goddard, 1991; Butterwith et al., 1992, 1993).

Expression of mRNA for TGF-fls 1-3 has been detected in both proliferating (Burt et al., 1992) and differentiated preadipocytes (Weiner et al., 1989; Burt et al., 1992), and TGF-fl protein has been detected in porcine adipose tissue (Richardson et al., 1989). In the 3T3-L1 preadipocyte, TGF-fl expression is reduced during differentiation. Whether this change is involved in the regulation of differentiation by an autocrine mechanism is not known. The TGF-/~ receptor is also down-regulated during differentiation in the 1246 preadipocyte cell line and in primary rat adipocyte precursors (Serrero and Mills, 1991a). This appears not to be the case in all preadipocyte cell lines and primary precursors, as some of them can still respond to TGF-fl even when fully differentiated (Torti et al., 1989; Dani et al., 1990; Butterwith and Gilroy, 1991). Whether TGF-fls 1-3 perform different regulatory functions within adipose tissue is not known.

A number of recent findings has provided strong evidence for a role of epidermal growth factor (EGF) and TGF-~ in the regulation of preadipocyte proliferation and differentiation. TGF-ct has been shown to stimulate proliferation of both chicken and mouse adipocyte precursors and to inhibit preadipocyte differentiation (Serrero, 1987; Butterwith et al., 1992). EGF, which acts through the same receptor as TGF-~, has similar effects (Serrero, 1987; Butterwith et al., 1992). Treatment of rats with EGF causes a reduction in the number of adipocytes and an increase in the number of adipocyte precursors, leading to a reduction in fat pad weight (Serrero and Mills, 1991b). This in vivo response is consistent with that observed in vitro.

It is not known, at present, whether EGF or TGF-~, has an autocrine/paracrine or endocrine action in adipocyte development. TGF-~, protein has been shown to be present in adipose tissue (Crandall et al., 1992), and Luetteke et al. (1993) have demonstrated expression of TGF-~t, and EGF receptor in a pooled sample of epididymal and renal fat pads prepared from mice. Serrero et al. (1993) failed to detect EGF expression in mouse adipose tissue using Northern blotting. This observation, coupled with depressed levels of plasma EGF in ob/ob mice compared with their control littermates, indicates a possible endocrine role for EGF in the regulation of adipocyte development (Serrero et al., 1993).

Overexpression of TGF-~ under control of a metallothionein promoter in transgenic mice leads to a 40-80% reduction in epididymal fat and a 50% reduction in total body fat. This is consistent with the results obtained by Serrero and Mills (1991b), who injected EGF into rats. In the latter work, other expected effects of EGF/TGF-~, were seen, such as accelerated eyelid opening and precocious incisor eruption. These effects were not seen in metallothionein-TGF-~, transgenic mice, suggesting that TGF-~, was acting in an autocrine/paracrine fashion in these animals.

A limited number of studies have looked at the effect of platelet-derived growth factor (PDGF) on preadipocyte proliferation and differentiation in vitro. PDGF stimulates proliferation in a number of preadipocyte cell lines (Aoki et al., 1990) and in primary cultures of adipocyte precursor cells (Butterwith and Goddard, 1991). Early evidence suggested that PDGF could inhibit preadipocyte differentiation (Hayashi et al., 1981). However, these experiments were done with PDGF purified from platelets which are also a source of TGF-/L These experiments, therefore, should be repeated using recombinant PDGF, which is now available. There is currently no information on whether PDGF is expressed in preadipocytes in culture or in adipose tissue in vivo.

Growth factor expression in adipocytes or their precursors may also regulate the functions of surrounding cells that are not of the adipocyte lineage. For example, 3T3-F442A preadipocytes

406 S.C. BUTTERWITH

have been shown to express vascular endothelial cell growth factor in a differentiation-specific manner (Claffey et al., 1992). This growth factor is specific for endothelial cells and is likely to play a role in the development of the vasculature within adipose tissue. Previous reports have shown a relationship between the development of blood vessels and the development of adipocytes (Hausman and Kauffman, 1986), and production of vascular endothelial cell growth factor by differentiating adipocytes might provide a signalling mechanism for this relationship. Monobutyrin, a vasoactive lipid secreted by adipocytes, is also likely to be involved (Wilkison et al., 1991; Wilkison and Spiegelman, 1993).

Early studies on the wasting condition termed cachexia showed marked reductions in body fat and muscle, and one of the mediators of the reduction in body fat was identified as TNF-~ (Torti et al., 1985; Beutler et al., 1985). Administration of TNF-ct, to a number of preadipocyte cell lines inhibits the differentiation process and is also able to induce dedifferentiation of fully differentiated cells. A number of other cytokines/growth factors have since been shown to have similar effects (Beutler and Cerami, 1985; Patton et al., 1986; Gregoire et al., 1992). It is likely that a number of these factors have a role in altering energy balance during infection and do not have a major role in the normal development of adipose tissue. Recent studies, however, suggest a role for increased production of TNF-ct by adipocytes in a number of acquired and genetic models of obesity. Expression of TNF-at is increased in adipose tissue in these obese strains compared with their lean counterparts (Hotamisligil et al., 1993).

Studies on the role of growth factors in the regulation of preadipocyte proliferation and differentiation are still at an early stage. Preadipocytes clearly respond to a number of growth factors in vitro, indicating the presence of specific receptors, but there have been few studies to date that have demonstrated the presence of receptors in vivo. A few studies have also demonstrated expression of mRNA for a number of growth factors in vitro, but there is very little information detailing in vivo expression. In particular, it will be important to determine which cell types in vivo are expressing growth factors, whether there are developmental changes in the pattern of expression and whether these correlate with adipocyte development. Assessment of the role of growth factors is further complicated by the presence of binding proteins, which regulate their action. This is particularly important in the case of the IGFs where six specific binding proteins have been identified so far. Growth factor activity is also regulated at the level of availability. For example, the FGFs can be sequestered in the extracellular matrix of a number of tissues and, therefore, even when present, may not be active. Thus, the presence of a growth factor at the mRNA or protein level does not necessarily signify an important action.

6. THE ROLE OF PROSTAGLANDINS AS AUTOCRINE/PARACRINE-ACTING REGULATORS OF ADIPOCYTE DEVELOPMENT

It has been known for sometime that adipose tissue is a site of prostaglandin production (Shaw and Ramwell, 1968; Christ and Nugteren, 1970). Preparation of an adipocyte suspension after collagenase digestion of adipose tissue showed that the adipocyte was a source of both prostacyclin and prostaglandin E2 (PGE2). However, it is very difficult, even with repeated washing, to prepare an adipocyte fraction that is totally free of other cell types. Parker et al. (1989) recently have proposed that it is vascular endothelial cells that produce prostacyclin in response to catecholamines and that adipocytes provide the arachidonic acid precursor.

Prostaglandins and, in particular, PGE2 have been shown in a number of studies to have marked inhibitory effects on adipose tissue lipolysis (Richelsen, 1992), but recent evidence suggests that they may also be important paracrine/autocrine regulators of preadipocyte differentiation. Arachidonic acid promotes the conversion of preadipocytes to adipocytes, possibly through an increase in the levels of cAMP, increased breakdown of inositol phospholipids (Gaillard et al., 1989) and an increase in intracellular calcium (Vassaux et aL, 1992). Furthermore, two metabolites of arachi- donic acid, prostacyclin and prostaglandin F2~ (PGF2~), are also able to stimulate preadipocyte differentiation in the Ob1771 preadipocyte cell line. This is in contrast to the parent cell Ob17, in which PGF2~-inhibited adipose conversion (Negrel et al., 1981). The latter study was performed in serum-containing medium, and it is possible that a serum factor, or factors, may modulate the

Molecular events in adipocyte development 407

response to PGF2~. This explanation seems unlikely as, Serrero et al. (1992) have investigated the effects of PGF2~ in serum-free medium, but in a different preadipocyte cell line. They found that PGF2~ was an inhibitor of both early and late differentiation markers. The inhibition was concentration dependent and had a similar effect in primary cultures of rat adipocyte precursors. The degree of inhibition was dependent on the timing of addition. Much less of an effect was seen when PGF2~ was added for the period during cell proliferation or after differentiation had been initiated. Evidence from in vivo studies also supports a negative role for prostaglandins in the regulation of preadipocyte differentiation. Prostaglandin production in obese Zucker rats is low compared with their lean counterparts (Gaskins et al., 1989).

At present, it is unclear whether PGF2~ is acting at a specific stage of the differentiation process, as proposed by Serrero et al. (1992), or whether this is a characteristic of the particular cell systems used. A similar phenomenon was seen with TGF-fl, which was shown to inhibit 3T3 cell differentiation only at an early stage (Ignotz and Massague, 1985; Sparks and Scott, 1986; Sparks et al., 1992), but in other cell lines and strains inhibited at all stages of differentiation (Torti et al., 1989; Dani et al., 1990; Butterwith and Gilroy, 1991). It is also unclear why PGF2~ has opposite effects in different cell lines.

The rate-limiting step in the production of eicosanoid mediators is catalyzed by PLA2. Gao and Serrero (1990) have shown that PLA2 activity is low in undifferentiated preadipocytes and rises 20- to 24-fold during differentiation. This increase in PLA2 activity correlated with a large increase in the level of PGF2~, but the level of PGE2 was unchanged between differentiated and undifferentiated cells. This provides further evidence for the mature adipocyte being a source of prostaglandins and the production of PGF2~ may provide a mechanism whereby differentiating cells could modulate the differentiation of other cells present in the precursor pool.

7. CONCLUSIONS AND FUTURE DIRECTIONS

Over the last 10 years, the use of in vitro cell culture systems has provided a great deal of information about the processes and molecular mechanisms that are involved in preadipocyte proliferation and differentiation and how these might be regulated. There are still, however, a number of gaps in our knowledge. The most fundamental involves the commitment of cells to the adipocyte lineage. Some progress has been made, recently, in this area (Graves et al., 1992; Colon-Teicher et al., 1993), and studies on transcriptional regulators of adipocyte-specific genes and DNA transfection studies, similar to that described by Colon-Teicher et al. (1993), should provide more information over the next few years. It is likely that master regulatory genes exist for adipose tissue, which play a similar role to those of the Myo D family in muscle commitment.

By studying the aP2 gene, a number of important DNA elements have been identified that are involved in differentiation-specific expression, and future investigations are likely to focus on which factors bind to these elements and how they interact. Specific studies on other differentiation- specific genes will help to identify any common regulatory mechanisms. The aP2 promoter studies have shown the importance of in vivo experiments using transgenic animals to complement in vitro studies when attempting to study the function of regulatory elements. The study of differentiation- specific genes in other species besides the mouse would assess whether these regulatory mechanisms are conserved across species or whether different mechanisms have evolved.

A number of questions still remain as to the precise role of growth factors in regulating adipocyte development in vivo. Very little is known about the expression of these factors and their receptors in adipose tissue and whether they have endocrine or autocrine/paracrine actions. Growth factors, such as TGF-fl, EGF/TGF-~, and TNF-~, have been shown in vitro to inhibit preadipocyte differentiation, but the mechanism of action is unknown. It is possible that some growth factors might regulate the expression of important transcription factors. TNF-~t has been shown to repress the expression of C/EBP in 3T3-L1 adipocytes (Stephens and Pekala, 1991, 1992). The mechanism of action of TGF-fl, EGF/TGF-~ awaits further study. It will also be of interest to see if any of the transcription factors that bind to the enhancer are under hormonal or growth factor control. Further studies are needed in these areas.

408 S.C. BUTTERWITH

The prostaglandins are another important group of autocrine/paracrine regulators of adipocyte development. Prostaglandins are produced by cells present in adipose tissue, and it is likely that there is a complex interaction between different cells and their prostaglandin production, which acts to regulate the differentiation of the precursor pool. Whether there is also an interaction with locally produced growth factors that may be acting in a similar fashion remains to be investigated.

The link between adipose tissue and the immune system is an intriguing one. Although the function of adipsin has been defined in vitro as a complement factor D activity, its precise function in vivo and why it is secreted from adipose tissue is not known. Similarly, the role of overexpression of TNF-oq in various models of acquired and genetic obesity is not known. New approaches using gene targeting should help in addressing these questions.

Acknowledgements--Work in the author's laboratory is supported by a commission from the Ministry of Agriculture, Fisheries and Food.

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