PHYSIOLOGICAL REVIEWS   Vol. 78 No. 3 July 1998, pp. 783-809
Copyright ©1998 by the American Physiological Society

Understanding Adipocyte Differentiation

FRANCINE M. GREGOIRE, CYNTHIA M. SMAS, AND HEI SOOK SUL

Department of Nutritional Sciences, University of California, Berkeley, California

I. INTRODUCTION
II. ADIPOSE TISSUE AS A SECRETORY ORGAN
III. ORIGIN OF ADIPOSE CELLS AND ADIPOSE TISSUE
IV. IN VITRO MODELS OF ADIPOCYTE DIFFERENTIATION
V. PROCESS OF ADIPOCYTE DIFFERENTIATION
    A. Growth Arrest
    B. Clonal Expansion
    C. Early Changes in Gene Expression
    D. Late Events and Terminal Differentiation
VI. FACTORS THAT MODULATE ADIPOCYTE DIFFERENTIATION
    A. Hormones and Signal Transduction Pathways Regulating Adipocyte Differentiation
    B. Pref-1, an EGF Repeat-Containing Inhibitor of Adipocyte Differentiation
    C. Extracellular Matrix Components
VII. TRANSCRIPTION FACTORS CRITICAL FOR ADIPOCYTE DIFFERENTIATION
    A. PPAR Family
    B. C/EBP Family
    C. bHLH Family
VIII. CONCLUSIONS
REFERENCES

	ABSTRACT

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Gregoire, Francine M., Cynthia M. Smas, and Hei Sook Sul. Understanding Adipocyte Differentiation. Physiol. Rev. 78: 783-809, 1998.  The adipocyte plays a critical role in energy balance. Adipose tissue growth involves an increase in adipocyte size and the formation of new adipocytes from precursor cells. For the last 20 years, the cellular and molecular mechanisms of adipocyte differentiation have been extensively studied using preadipocyte culture systems. Committed preadipocytes undergo growth arrest and subsequent terminal differentiation into adipocytes. This is accompanied by a dramatic increase in expression of adipocyte genes including adipocyte fatty acid binding protein and lipid-metabolizing enzymes. Characterization of regulatory regions of adipose-specific genes has led to the identification of the transcription factors peroxisome proliferator-activated receptor- (PPAR-) and CCAAT/enhancer binding protein (C/EBP), which play a key role in the complex transcriptional cascade during adipocyte differentiation. Growth and differentiation of preadipocytes is controlled by communication between individual cells or between cells and the extracellular environment. Various hormones and growth factors that affect adipocyte differentiation in a positive or negative manner have been identified. In addition, components involved in cell-cell or cell-matrix interactions such as preadipocyte factor-1 and extracellular matrix proteins are also pivotal in regulating the differentiation process. Identification of these molecules has yielded clues to the biochemical pathways that ultimately result in transcriptional activation via PPAR- and C/EBP. Studies on the regulation of the these transcription factors and the mode of action of various agents that influence adipocyte differentiation will reveal the physiological and pathophysiological mechanisms underlying adipose tissue development.

	I. INTRODUCTION

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White adipose tissue (WAT) is the major energy reserve in higher eukaryotes, and storing triacylglycerol in periods of energy excess and its mobilization during energy deprivation are its primary purposes. Recently, there has been a dramatic increase in the incidence of obesity resulting from an excess of WAT. Obesity is a prevalent health hazard in industrialized countries (145, 298) and is closely associated with a number of pathological disorders, including non-insulin-dependent diabetes, hypertension, cancer, gallbladder disease, and atherosclerosis. With regard to this wide range of health implications, the need to develop new and effective strategies in controlling obesity has become more acute. Recently, progress has been made in understanding the process of adipocyte differentiation and in the cloning of genes mutated in several monogenic mouse models of obesity. This has not only allowed us to begin to understand the cellular and molecular basis of adipose tissue growth in physiological and pathophysiological states, but has also provided means to develop therapeutic strategies for the treatment and prevention of obesity. This review addresses regulation of WAT development at the cellular and molecular levels. We summarize information gained from studies in preadipocyte cell lines and attempt to incorporate results from often neglected primary preadipocyte studies.

	II. ADIPOSE TISSUE AS A SECRETORY ORGAN

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Mature adipocytes, the main cellular component of WAT, are uniquely equipped to function in energy storage and balance under tight hormonal control. However, with the recent realization that adipocytes secrete factors known to play a role in immunological responses, vascular diseases, and appetite regulation, a much more complex and dynamic role of WAT has progressively emerged. Leptin, the obese (ob) gene product, is a hormone that is primarily made and secreted by mature adipocytes and binds to its receptor in the hypothalamus. Studies indicate leptin may function in regulating body fat mass. Loss of fat stores decreases leptin levels and increases neuropeptide Y levels; this leads to increased food intake. Conversely, weight gain increases leptin levels leading to decreased food intake; melanocyte-stimulating hormone is necessary for this response (31, 201). Leptin levels are elevated in human obesity and in animal models of obesity. More recently, the leptin receptor has been detected in peripheral tissues. This suggests additional roles for leptin, including modulation of insulin action in liver (45), production of steroids in the ovary (310), and direct effects on adrenocortical steroidogenesis (23). Leptin also has a role in reproductive physiology (39, 178) and is involved in hematopoietic and immune system development (18, 173). This rapidly growing list, clearly not exhaustive, indicates that leptin has a much broader range of action than initially perceived.

Immune system-related proteins produced by adipocytes include adipsin, acylation stimulation protein (ASP), adipocyte complement-related protein (Acrp30/AdipoQ), tumor necrosis factor- (TNF-), and macrophage migration inhibitory factor (MIF). With the exception of TNF-, their physiological function remains to be elucidated. Nonetheless, these adipocyte-derived factors might also be involved in either the control of energy homeostasis or insulin resistance. Acylation stimulation protein is generated by the interaction of complement factor D (identical to adipsin), factor B, and complement C3, components of the alternate complement pathway (41); ASP may be involved in regulating energy storage by stimulating triacylglycerol synthesis and glucose transport (44, 168, 252). Acrp30/AdipoQ displays sequence homology with C1q, the first component of the classical complement activation pathway. Like adipsin, Acrp30/AdipoQ is abundant in normal serum, and its secretion is enhanced by insulin. This suggests that it could function as a signaling molecule from adipocytes and might therefore regulate energy homeostasis (119, 229). As addressed in detail in section VIA, TNF- not only inhibits adipocyte differentiation, but treatment of mature adipocytes with TNF- reduces the expression of adipocyte genes (202, 259, 260, 273, 274). In addition, TNF- may contribute to the insulin resistance that accompanies obesity and non-insulin-dependent diabetes mellitus; TNF- levels are elevated in WAT of obese rodents and humans. This may contribute to insulin resistance by inhibiting insulin-stimulated tyrosine kinase activity of the insulin receptor (114-117, 135). Treatment of adipocytes with TNF- was recently reported to increase secretion of MIF, a proinflammatory cytokine. Therefore, MIF may mediate the above-mentioned contribution of TNF- to insulin resistance (112).

Vascular function-related proteins that are secreted by adipocytes include angiotensinogen and plasminogen activator inhibitor type 1 (PAI-1). White adipose tissue contains all the main components of the renin-angiotensin system such as angiotensinogen, angiotensin converting enzyme, angiotensin II, and angiotensin receptors (130). Angiotensinogen could play a role in regulating adipose tissue blood supply and fatty acid efflux from fat (73). Angiotensin II, the cleavage product of angiotensinogen, has been implicated in adipose tissue growth by stimulating production of prostacyclin by mature fat cells and thereby promoting adipocyte differentiation via a paracrine/autocrine mechanism (53). Because angiotensin II increases lipogenesis in both human and 3T3-L1 adipocytes, it may also be involved in the control of adiposity through regulation of lipid synthesis and storage in adipocytes (127). Plasminogen activator inhibitor type 1 is produced by adipose tissue, and treatment of 3T3-L1 adipocytes with transforming growth factor- (TGF-) significantly increases PAI-1 production. Higher PAI-1 levels have been reported in omental fat compared with subcutaneous fat. This may be correlated with increased PAI-1 levels noted in central obesity and may be involved in the development of vascular diseases associated with abdominal obesity (5, 164, 241).

Taken together, these studies clearly establish that the adipocyte behaves as an endocrine as well as a paracrine/autocrine cell. Along with its active role in regulating energy balance, WAT has the potential to play a dynamic role in a variety of other physiological processes, including the autoregulation of adipose tissue growth and development.

	III. ORIGIN OF ADIPOSE CELLS AND ADIPOSE TISSUE

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The origins of the adipose cell and adipose tissue are still poorly understood, and the molecular events leading to the commitment of the embryonic stem cell precursor to the adipocyte lineage remain to be characterized. In most species, WAT formation begins before birth, as assessed by morphological studies performed on human, pig, mouse, and rat embryos (59, 204, 205, 245). The chronology of WAT appearance, however, is strictly dependent on the species as well as the adipose depot (59, 218, 245). White adipose tissue expansion takes place rapidly after birth as a result of increased fat cell size as well as an increase in fat cell number. Even at the adult stage, the potential to generate new fat cells persists. It has been demonstrated that fat cell number can increase when rats are fed a high-carbohydrate or high-fat diet (67, 68, 176). Increase in fat cell number is also observed in severe human obesity. However, the relative contribution of fat cell size and fat cell number to human adipose tissue growth on nutritional stimulation remains to be clarified. Regardless, early differentiation markers of adipocyte differentiation can be detected even in adipose tissue derived from very old mice (138). Moreover, fat cell precursors isolated from adult WAT of various species, including humans, can be differentiated in vitro into mature adipocytes (21, 58, 98, 104, 160, 213, 264). The potential to acquire new fat cells from fat cell precursors throughout the life span is now undisputed.

Although the developmental origin of fat cells is not known, several studies on multipotent clonal cell lines have suggested that the adipocyte lineage derives from an embryonic stem cell precursor with the capacity to differentiate into the mesodermal cell types of adipocytes, chondrocytes, osteoblasts, and myocytes. Treatment of the murine embryonic cell line C3H10T1/2 with a demethylating agent generates loci of muscle, cartilage, and fat cells (143, 265); 25% of colonies contained myofibers, 7% adipocytes, and 1% chondrocytes. The C3H10T1/2 cells may represent multipotential stem cells that are blocked at the mesodermal pathway. That a single gene could convert C3H10T1/2 cells into myoblasts has been demonstrated with the identification and characterization of the MyoD family of regulatory genes (54). The lower clonal frequency of conversion of C3H10T1/2 cells to adipocytes as compared with myocytes may indicate that more genes are required to activate adipocyte development. However, these different frequencies of conversion may also somewhat reflect culture conditions that could selectively promote the differentiation of one cell lineage over another. There is also evidence that a common bone marrow stromal cell type may give rise to adipogenic or osteogenic cells with several indications of a reciprocal relationship in the differentiation of these two cell types (84). For example, bone morphogenetic proteins of the TGF- superfamily act as potent osteogenic agonists (83), whereas TGF- inhibits adipocyte differentiation, as discussed in section VIA. Furthermore, the antidiabetic compounds thiazolidinediones, recently discovered to act through peroxisome proliferator-activated receptor- (PPAR-), an adipogenic transcription factor described in section VIIA, reduce bone marrow density and increase bone marrow adipocytes (84). Teratocarcinoma-derived C1 cells also behave as progenitor cells with osteoblast, chondroblast, or adipoblast cell fates (206). This suggests a close ontogenetic relationship between these connective tissue cell types. Osteogenic, chondrogenic, and adipogenic cells may arise from sclerotomal cells. The recent cloning of the basic-helix-loop-helix (bHLH) transcription factors twist and scleraxis has provided hints about the initial events that might precede and/or lead to the formation of the adipose lineage. In mouse embryo, these genes appear to play a significant role in the development of mesodermal tissues. Twist is expressed in early somites, and its expression is restricted to the sclerotome and excluded from myotome upon somite compartmentalization. Twist may be essential for the establishment of mesodermal cell fate and may be involved in the subdivision of the mesoderm lineage later in development (85). Significant levels of scleraxis can only be detected after somite compartmentalization, when it is expressed in sclerotome but not in myotome. Scleraxis could be a regulator of gene expression within the mesenchymal cell lineages that give rise to connective tissues (50). The precise role of these genes in adipocyte determination remains to be established.

	IV. IN VITRO MODELS OF ADIPOCYTE DIFFERENTIATION

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For the past 20 years, in vitro systems have been extensively used to study adipocyte differentiation. This has led to a dissection of the molecular and cellular events taking place during the transition from undifferentiated fibroblast-like preadipocytes into a mature round fat cells. Various preadipose cell lines and primary cultures of adipose-derived stromal vascular precursor cells have been used. Table 1 summarizes the characteristics of the most commonly employed preadipose cell models. Preadipose cell lines as well as primary preadipocytes are already committed solely to the adipocyte lineage, although they may represent different stages of adipocyte development. The most frequently employed cell lines are 3T3-F442A and 3T3-L1. These were clonally isolated from Swiss 3T3 cells derived from disaggregated 17- to 19-day mouse embryos (89, 90, 92). 3T3-C2 cells derive from the same source but are not preadipocyte in nature. They do not differentiate into adipocytes and can be used to compare the responses of differentiation-defective with differentiation-competent cell types. The TA1 cell line was established by treating CH310T1/2 mouse embryo fibroblast cells with the demethylating agent 5-azacytidine (37, 143, 265). Ob17 cells and their derivatives were generated from adipose precursors present in the epididymal fat pads of genetically obese (ob/ob) adult mice (187). In addition to these models, embryonic stem (ES) cells have recently been shown to differentiate into mature adipocytes in vitro (52) and will likely provide a useful system for investigating the initial steps of adipogenesis. In vitro-differentiated adipocytes have many characteristics of adipose cells in vivo. Subcutaneous injection of preadipose cells in nude mice leads to development of mature fat pads that are histologically indistinguishable from WAT; this is convincing evidence that adipose cell acquisition occurs by a similar mechanism in vivo (91, 278).

Because the stage of differentiation and the lineage of preadipocyte cell lines have not been well established, primary cultures have been particularly useful for validating results obtained in preadipose cell lines. Primary preadipocytes have been successfully cultured from a number of species including humans and have several advantages over preadipose cell lines (96-98, 104, 106, 138, 160, 191, 236). Primary cells are diploid and may therefore reflect the in vivo context better than aneuploid cell lines. Second, they can be derived from adipose tissue obtained from various species at different postnatal stages of development and from various adipose depots (21, 57, 58). The latter is of particular interest, since molecular and biochemical differences in fat pads have been observed (96, 150, 151, 169, 212, 308). Such differences may have major physiological consequences, since excessive centralized fat accumulation (upper body obesity) is associated with increased morbidity in humans (139). On the other hand, the potential cellular heterogeneity of the stromal vascular preadipose cell population is a drawback of primary cultures. This, however, may be a minor concern, since nearly 100% differentiation can be achieved when preadipocytes derived from young animals are adequately treated (57, 97, 236). For primary preadipocyte cultures, differentiation capacity is clearly donor dependent and decreases significantly with age (21, 58, 98, 138). The molecular basis for this reduced differentiation capacity is not known. It may, however, indicate that preadipose cells derived from aged donors represent a subpopulation of precursor cells arrested at distinct stage(s) of adipocyte development. These cells may therefore require a different subset of as yet to be characterized signals to undergo adipocyte differentiation (63, 98).

During the growth phase, cells of preadipocyte lines as well as primary preadipocytes are morphologically similar to fibroblasts. At confluence, induction of differentiation by appropriate treatment leads to drastic cell shape changes. The preadipocyte converts to a spherical shape, accumulates lipid droplets, and progressively acquires the morphological and biochemical characteristics of the mature white adipocyte. The nature of the induction is dependent on the specific cell culture model system employed. A variety of differentiation protocols have been developed for preadipose cell lines and for primary preadipocytes. Embryonic stem cells can be differentiated into adipocytes at high efficiency in vitro if early-developing ES cell-derived embryoid bodies are exposed to retinoic acid (RA) for a precise time, followed by treatment with standard adipogenic hormones (52). 3T3-L1 preadipocytes spontaneously differentiate over a period of several weeks into fat-cell clusters when maintained in culture with fetal calf serum. This can be accelerated by the inducing agents dexamethasone and methylisobutylxanthine (MIX), a phosphodiesterase inhibitor. High concentrations of insulin have been used in combination with these inducing agents. The identification of agents that accelerate differentiation, but that are not required to maintain the differentiated phenotype, provides insights to the biochemical pathways that may function during adipocyte differentiation.

Although preadipocytes from various sources are similar in many regards, their responsiveness to inducing agents varies considerably. This may represent differences in the stage of maturation at which the preadipocytes were originally harvested during cloning of cell lines or the age and/or source of tissue from which primary cells are isolated. The effects of the various inducing agents on adipocyte differentiation are discussed in detail in section VI. In most cases, primary preadipocytes derived from young animals require only either low concentrations (1-10 nM) of insulin in the presence of fetal calf serum, or high concentrations (1-10 µM) in serum-free medium (57, 97, 104, 264). Depending on the species, the age of the donor, and/or the adipose depot source, agents such as glucocorticoids and MIX are either necessary to trigger the differentiation program or act only to accelerate it (57, 58, 96, 97, 104, 264, 293, 309). For established cell lines as well as for primary cells, the development of serum-free culture conditions has confirmed the positive role of these inducers and the involvement of insulin-like growth factor I (IGF-I), glucocorticoid, and cAMP signaling pathways. For preadipose cell lines, addition of insulin at supraphysiological concentrations does not affect the number of differentiated cells but does serve to accelerate lipid accumulation. In contrast, for most primary preadipocytes, which have a very low degree of spontaneous differentiation, insulin increases the number of differentiated cells; few lipid-containing cells are observed in the absence of insulin (95, 104, 264). Depending on the culture system, the insulin/IGF-I signaling pathway may either be critical for differentiation or necessary only to achieve the maximal rate of triacylglycerol accumulation that accompanies adipocyte differentiation.

	V. PROCESS OF ADIPOCYTE DIFFERENTIATION

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An overview of the stages of adipocyte differentiation is presented in Figure 1. The committed preadipocyte maintains the capacity for growth but has to withdraw from the cell cycle before adipose conversion. During adipocyte differentiation, acquisition of the adipocyte phenotype is characterized by chronological changes in the expression of numerous genes. This is reflected by the appearance of early, intermediate, and late mRNA/protein markers and triglyceride accumulation. These changes take place primarily at the transcriptional level, although posttranscriptional regulation occurs for some adipocyte genes (180, 295). In addition to the activation of genes, those genes that are inhibitory to adipogenesis or simply unnecessary for adipose cell function are repressed. Changes in gene expression during the early and late stages of adipocyte maturation have been characterized mainly through the use of preadipose cell lines. These have been extensively reviewed by others (47, 166, 249) and are summarized below. The limited information that is available on the details of the molecular events occurring during primary preadipocyte differentiation is also incorporated.

View larger version (32K): [in this window] [in a new window]   FIG. 1.   Overview of stages in adipocyte differentiation. Our current understanding of adipocyte differentiation indicates that a pluripotent stem cell precursor gives rise to a mesenchymal precursor cell with the potential to differentiate along mesodermal lineages of myoblast, chondroblast, osteoblast, and adipocyte. As discussed in text, given appropriate environmental and gene expression cues, preadipocytes undergo clonal expansion and subsequent terminal differentiation. Selected molecular events accompanying this process are indicated to right, with their approximate duration reflected by the solid line. PPAR-, peroxisome proliferator-activated receptor-; C/EBP, CCAAT/enhancer binding protein; pref-1, preadipocyte factor-1; ECM, extracellular matrix; FA, fatty acid.

In preadipose cell lines as well as in primary preadipocytes, growth arrest and not cell confluence or cell-cell contact per se appears to be required for adipocyte differentiation. Confluent 3T3-F442A cells shifted to methylcellulose-stabilized suspension culture still undergo differentiation (196). This indicates that although confluence in these cells leads to growth arrest, cell-cell contact is not a prerequisite for adipocyte conversion. Primary rat preadipocytes plated at low density in serum-free medium can also differentiate in the absence of cell-cell contact (277). Two transcription factors, CCAAT/enhancer binding protein  (C/EBP-) and PPAR-, have been shown to transactivate adipocyte specific genes, and are discussed in section VII. Both C/EBP- and PPAR- also appear to be involved in the growth arrest that is required for adipocyte differentiation. McKnight and co-workers (276) have demonstrated the antimitotic activity of C/EBP- through the use of a C/EBP--estrogen receptor fusion protein. Activation by estrogen treatment results in cessation of cell growth as assessed by cell number and DNA synthesis (276). Darlington and co-workers (268) have reported that C/EBP- increases p21/SDI-1 mRNA and protein levels, but not other cell cycle components. Moreover, antisense p21/SDI-1 eliminates growth inhibition brought about by C/EBP- (268). This indicates that C/EBP- may function by increasing p21/SDI-1 levels. Spiegelman and colleagues (6) have showed that PPAR- is sufficient to induce growth arrest in fibroblasts and in adipogenic simian virus 40 large T antigen-transformed cells. In these PPAR--expressing cells, cell cycle withdrawal is accompanied by a decrease in the DNA binding and transcriptional activity of the E2F/DP-1 complex because of phosphorylation of DP-1 and a decrease in the expression of the serine/threonine phosphatase PP2A catalytic subunit. Therefore, C/EBP- and PPAR- may act cooperatively to bring about growth arrest (6). Although C/EBP- and PPAR- expression increases dramatically during adipocyte differentiation, the low level of these factors expressed in preadipocytes may be sufficient to mediate growth arrest that precedes differentiation.

After growth arrest at confluence, preadipocytes must receive an appropriate combination of mitogenic and adipogenic signals to continue through subsequent differentiation steps. Studies on preadipose cell lines have shown that growth-arrested cells undergo at least one round of DNA replication and cell doubling. This has been proposed to lead to the clonal amplification of committed cells (196). For 3T3-F442A and Ob17 cells, an increase in DNA synthesis precedes expression of late mRNA markers, and inhibition of DNA synthesis prevents the formation of fat cells (9, 147). However, primary preadipocytes derived from human adipose tissue do not require cell division to enter the differentiation process (63). In these cells, inhibition of mitosis with cytosine arabinoside does not impair adipocyte development, indicating that clonal amplification of committed cells is not a critical step. These cells may have already undergone potential critical cell divisions in vivo and may therefore correspond to a later stage of adipocyte development.

Examination of growth-related proteins indicates potential differences between clonal expansion and preconfluent cell growth in 3T3-L1 preadipocytes. Retinoblastoma proteins pRB, p107, and p130 bind the E2F/DP complex to inactivate growth-promoting transcriptional activities. A recent report indicates changes in the expression of retinoblastoma proteins during differentiation of 3T3-L1 cells, including a transient increase in p107. This appears specific to clonal expansion, since this change is not detected during serum-stimulated cell growth (217). The role of E2F and RB family members during adipogenesis is unknown and needs further examination. Similarly, another group of growth arrest-specific (gas) genes shows a distinct expression pattern during clonal expansion. Gas6 appears to be preferentially expressed during clonal expansion of postconfluent preadipocytes, whereas gas1 and gas3 are expressed in serum-starved preadipocytes (244). Combined, these observations suggest differential regulation of the cell cycle in preconfluent proliferation versus postconfluent hormonally stimulated clonal expansion. An intracellular receptor for the immunosuppressive drug FK506, FKBP51, also transiently accumulates during the clonal expansion phase. Interestingly, whereas FK506 itself does not have any effect on clonal expansion and adipocyte differention, at high concentrations it reverses the inhibitory effect of rapamycin, another immunosuppressant that shares intracellular receptors (FKBPs) with FK506 (305, 307) Regardless of its mechanism, inhibition of proliferation by rapamycin is accompanied by inhibition of mitogen-activated serine-threonine S6 kinase, indicating that clonal expansion is necessary for subsequent adipocyte differentiation of 3T3-L1 cells. This also suggests that a phosphorylation-dephosphorylation mechanism may be involved during the clonal expansion period. Interestingly, a transient increase in levels of the phosphatase inhibitor HA2 occurs during the clonal expansion phase. Constitutive expression of HA2 blocks differentiation only during the clonal expansion period but not during later stages of differentiation. Moreover, inhibition of adipocyte differentiation by HA2 can be overcome by treatment with the phosphatase inhibitor vanadate, suggesting that a critical tyrosine dephosphorylation is necessary during clonal expansion (157).

Although it is helpful to schematize the stages of adipocyte differentiation into a hierarchy of molecular events, an accurate chronology of the earliest steps in adipocyte differentiation has not been elucidated. Growth arrest and clonal expansion are accompanied by complex changes in the pattern of gene expression that can differ with the cell culture models and the specific differentiation protocols employed. Expression of lipoprotein lipase (LPL) mRNA has often been cited as an early sign of adipocyte differentiation (3, 47, 98, 123, 166). Lipoprotein lipase is secreted by mature adipocytes and plays a central role in controlling lipid accumulation (48, 86). However, LPL expression occurs spontaneously at confluence and is independent of the addition of agents required for adipocyte differentiation (8, 9, 277). This suggests that LPL expression may reflect the growth-arrest stage rather than being an early differentiation step. It is also synthesized and secreted by other mesenchymal cell types including cardiac muscle cells and macrophages (49, 267). Because LPL expression is not adipocyte specific and it is independent of the additional agents required for adipocyte differentiation, classification of LPL as an early marker of adipocyte differentiation remains somewhat questionable.

At least two families of transcription factors, C/EBP and PPAR, are induced early during adipocyte differentiation. The early expression of C/EBP and PPAR is logical given their subsequent involvment in terminal differentiation by transactivation of adipocyte-specific genes, described in section VII. Peroxisome proliferator-activated receptor- is largely adipocyte specific and is expressed at low but detectable levels in preadipocytes. Its expression rapidly increases after hormonal induction of differentiation. It is easily detectable during the second day of 3T3-L1 adipocyte differentiation, and maximal levels of expression are attained in mature adipocytes (27, 38). Induction of PPAR- appears to precede that of PPAR-. Expression of PPAR-, however, is rather widespread. It is detected in a variety of tissues as well as in several cultured cell lines, including the CH310T1/2, 3T3-C2, and NIH 3T3 (7). A transient increase in the expression of C/EBP- and C/EBP- isoforms precedes the increase in PPAR- expression (27, 167, 299). The subsequent decrease of C/EBP- and C/EBP- in early to mid stages of differentiation is concomitant with the induction of C/EBP- mRNA. This increase in C/EBP- expression occurs slightly before the expression of adipocyte-specific genes (27, 153, 167). Another transcription factor induced very early during adipocyte differentiation is sterol regulatory element binding protein-1c (SREBP-1c)/adipocyte determination and differentiation factor 1 (ADD1), a bHLH-leucine zipper protein that is involved in cholesterol metabolism (26) and may also participate in adipocyte gene expression (136, 137).

During adipocyte differentiation, cells convert from a fibroblastic to a spherical shape, and dramatic changes occur in cell morphology, cytoskeletal components, and the level and type of extracellular matrix (ECM) components. Many of the studies on the effect of cytoskeletal and ECM components in adipocyte differentiation predate the characterization of adipocyte transcription factors. It is likely that these changes could influence the expression and action of PPARs and/or C/EBPs during adipocyte differentiation. Decrease in actin and tubulin expression is an early event in adipocyte differentiation that precedes overt changes in morphology and the expression of adipocyte-specific genes (255). These changes in cell shape reflect a distinct process in differentiation and are not the result of accumulated lipid stores. 3T3 preadipocytes can undergo biochemical and morphological differentiation even in conditions when triglyceride accumulation is blocked by deprivation of biotin, a cofactor for fatty acid synthesis (148), or by addition of lipolytic agents (258). A switch in collagen gene expression is also an early event of adipocyte differentiation. The relative concentrations of fibroblast-expressed type I and type III procollagen mRNA decline by 80-90% during 3T3-L1 differentiation, and secretion of type IV collagen and entactin/nidogen increases (13, 290). Expression of ₂-collagen type VI mRNA is first detectable upon confluence in Ob1771 cells and rises sharply after confluence (51). It reaches maximal levels 4 days postconfluence and gradually decreases to 50% of maximal levels during differentiation (51). Increased production of soluble and cell-associated chondroitin sulfate proteoglycan-I (versican) has been reported during 3T3-L1 differentiation, and this may account for the observed increase in culture medium viscosity (29). The amount of pericellular fibronectin, as well as cellular synthesis of fibronectin, decreases by four- to fivefold during differentiation of 3T3-F442A cells (10). Preadipocyte factor-1 (pref-1), a recently described preadipocyte protein with epidermal growth factor (EGF)-like repeats, discussed in section VIB, has been hypothesized to be involved in maintaining the preadipose phenotype (246-248). A dramatic decrease in pref-1 expression accompanies adipocyte differentiation; it is abundant in preadipocytes and is not detectable in mature fat cells. It is the only known gene whose expression is completely downregulated during adipocyte differentiation.

During the terminal phase of differentiation, adipocytes in culture markedly increase de novo lipogenesis and acquire sensitivity to insulin. The activity, protein, and mRNA levels for enzymes involved in triacylglycerol metabolism including ATP citrate lyase, malic enzyme, acetyl-CoA carboxylase, stearoyl-CoA desaturase (SCD1), glycerol-3-phosphate acyltransferase, glycerol-3-phosphate dehydrogenase, fatty acid synthase, and glyceraldehyde-3-phosphate dehydrogenase increase 10- to 100-fold (200, 256, 291). Glucose transporters (80), insulin receptor number, and insulin sensitivity increase. During adipocyte differentiation, there is a loss of ₁-adrenergic receptors and an increase in the ₂- and the ₃-subtypes; this results in an increase in total adrenergic receptor number (69, 70, 101, 152). In addition to increases in mRNAs for proteins directly related to lipid metabolism, adipocytes also synthesize other adipose tissue-specific products. These include the following: aP2, an adipocyte-specific fatty acid binding protein also identified as 422 (19, 256); FAT/CD36, a putative fatty acid transporter (125, 239); and perilipin, a lipid droplet-associated protein (94). In addition, adipocytes produce a number of secreted products, discussed in section II. These include the following: monobutyrin, an angiogenic agent; adipsin, a homolog of the serine protease complement factor D; Acrp30/AdipoQ; PAI-1; and angiotensinogen II (5, 41, 61, 119, 127, 229). Leptin is also increased during in vitro terminal differentiation of adipocytes, although its level is much lower than that detected in adipose tissue (165). Peroxisome proliferator activated receptor- and/or C/EBP- is implicated in the coordinate activation of several of these genes, including aP2, GLUT4, SCD1, phosphenolpyruvate carboxykinase (PEPCK), and leptin (113, 162, 175, 269, 270). The function of PPAR- and C/EBP- in adipocyte differentiation is presented in section VII.

Experiments with BALB/c 3T3 mesenchymal stem cells indicate that cells that have progressed beyond a specific stage in the differentiation process are committed to subsequent terminal differentiation and can neither dedifferentiate nor reenter mitosis (287, 294). However, recent evidence indicates that the precise stage beyond which adipocytes can be considered terminally differentiated is not clearly defined. Partially differentiated human preadipocytes, evidenced by substantial cytoplasmic lipid accumulation, are still capable of cell division as assessed histologically and by flow cytometry (210). The progressive dedifferentiation of primary mature adipocytes followed by cell division has also been reported (262). Tumor necrosis factor- treatment of mature TA1 or 3T3-L1 adipocytes or newly differentiated primary human adipocytes results in decreased expression of adipocyte markers and loss of lipid, with the development of morphological changes resulting in long, spindle-shaped cytoplasmic extensions (202, 273, 274). These cells therefore come to resemble preadipocytes. However, recent evidence indicates that although TNF--treated adipocytes and preadipocytes appear to share many similar morphological characteristics and gene expression patterns, they likely differ considerably. As described in section VIB, pref-1 expression is easily detected in preadipocytes and is absent from mature adipocytes; pref-1 levels are not restored by TNF- of mature adipocytes. Pref-1 expression may reflect a fundamental difference between naive preadipocytes and those that result from TNF- treatment (301). True reversion to a preadipose phenotype, if it takes place in vivo, could play a role regulating adipocyte number and consequently adipose tissue mass. The molecular and cellular events occurring during this process warrant more detailed study.

	VI. FACTORS THAT MODULATE ADIPOCYTE DIFFERENTIATION

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The growth and differentiation of animal cells are controlled by communication between individual cells or between cells and the extracellular environment. Adipocyte differentiation therefore requires the cell to process a variety of combinatorial inputs during the decision to undergo differentiation. Hormones and growth factors with a role in adipocyte differentiation act via specific receptors to transduce external growth and differentiation signals through a cascade of intracellular events. Identification of agents or molecules that modulate the process in either a positive or negative manner provides insight into the signal transduction pathways involved. Extracellular matrix proteins may play an important role in modulating adipocyte differentiation by permitting the morphological changes and adipocyte-specific gene expression that accompany differentiation. The combination of hormones and growth/differentiation factors that trigger or potentiate adipocyte differentiation has been extensively examined and is summarized in Table 2. All preadipose cell lines exhibit some degree of spontaneous conversion when grown in the presence of fetal calf serum, indicating serum is integral to the differentiation process. However, serum components are neither well characterized nor easily controllable. The development of chemically defined serum-free media suitable for the differentiation of preadipose cell lines and primary preadipocytes has aided the assessment of the precise hormonal requirements for differentiation. Although the full complement of inducing agents required for differentiation varies with each cell culture model, IGF-I, cAMP, and glucocorticoids are generally considered necessary for the induction of differentiation either in serum-containing or in serum-free media. In addition to stimulatory factors, agents that suppress the differentiation of preadipocytes have also been identified. These may act either by their mitogenic properties, as for various growth factors, or by other independent mechanisms, as in the case of pref-1. Recent work has begun to clarify how these exogenous signals may influence transcription factors such as C/EBP and PPAR- that are critical for activation of adipocyte genes and adipocyte differentiation.

2. Other growth factors and cytokines

Unlike IGF-I, other growth factors and cytokines are generally considered as inhibitors of adipocyte differentiation. This is perhaps because of the their mitogenic effects, since cell growth and differentiation are usually mutually exclusive. As discussed in section VA, growth arrest is requisite for differentiation. Several studies indicate a role for those growth factors that function through the EGF receptor, such as EGF and TGF-, in adipose tissue development. Transforming growth factor- inhibits differentiation of 3T3-F442A and rat preadipocytes, and transgenic mice overexpressing TGF- have a 50% reduction of total body fat (

4. Prostaglandins

Several lines of evidence indicate that arachidonate metabolites may play an important physiological role in adipose tissue metabolism and development. Mature adipocytes and cultured preadipocytes produce significant amounts of prostaglandins (PGs), including PGF₂ , PGE₂ , PGD₂ , and PGI₂ (

2

2

2

2

2

2

2

5. cAMP, G proteins, and protein kinase C

Methylisobutylxanthine accelerates the differentiation of preadipose cell lines and primary preadipocytes. As with glucocorticoids, MIX is routinely used for the differentiation of a variety of preadipocytes. Methylisobutylxanthine has been shown to increase expression of C/EBP-, and this increase is required for subsequent PPAR- expression and adipocyte differentiation. The role of C/EBP- in adipogenesis is presented in detail in section VII. The precise mode of action of MIX is not resolved. Methylisobutylxanthine is known to inhibit phosphodiesterases and block A₁ adenosine receptor in a competitive manner. It also stimulates adenylyl cyclase activity by blocking the inhibitory regulatory protein G_i (

Comparison of the genes that are regulated by the adipogenic inducing agents dexamethasone/MIX in 3T3-L1 cells and the closely related but differentiationdefective 3T3-C2 cells has resulted in the identification of preadipocyte factor-1 (pref-1). 3T3-C2 cells, which do not undergo differentiation in response to the adipogenic inducing agents dexamethasone/MIX, express approximately threefold higher pref-1 levels than 3T3-L1 preadipocytes. Recent evidence has demonstrated that pref-1 is a EGF repeat-containing transmembrane protein that inhibits adipocyte differentiation and suggests that this molecule may link adipocyte differentiation signals from the extracellular environment to the cell interior (246-248, 250). Expression of pref-1 in 3T3-L1 preadipocytes decreases to undetectable levels during their differentiation to mature adipocytes. Fetal calf serum, an essential component for in vitro adipocyte differentiation, dramatically downregulates pref-1 levels. Taken together, these observations indicate that pref-1 may not only be regulated during adipocyte differentiation, but suggest a regulatory role for pref-1 in adipocyte differentiation. Preadipocyte factor-1 does not appear to have the mitogenic effects (C. Li, C. M. Smas, and H. S. Sul, unpublished data) normally associated with the inhibitory actions of growth factors and cytokines described in section VIA2. This suggests that pref-1 may function via a unique mechanism to inhibit adipocyte differentiation.

Links between exogenous hormones and transcriptional regulators have begun to emerge, thereby allowing some integration of these two aspects in our understanding of the control of adipocyte differentiation. In contrast, knowledge of the molecular mechanisms regarding the effect of the ECM on adipocyte differentiation is lacking. These studies are particularly crucial if we are to fully understand the process in tissues, where differentiation occurs in close association with the stromal-vascular cell population. Cell adhesion molecules and ECM components modulate the interaction of cells with their environment in a manner that influences cell differentiation and migration. This may lead to cytoskeletal network rearrangement and an intracellular cascade of signal transduction that influences differentiation. Additionally, a physical connection between the ECM and nuclear matrix, via the cytoskeleton, has been proposed (129). The ECM, through altering cell spreading, may expose the cell surface to various growth factors present in the environment (181), and ECM components have been demonstrated to bind and sequester growth factors (303). The effect of ECM components on cell differentiation is demonstrated in general by the fact that a decrease in tissue-specific markers often occurs upon plating of primary cells onto tissue-culture dishes. Interaction between cell-matrix and nuclear events has been evidenced by numerous studies including -casein induction by basement membrane substratum of mammary epithelial cells (261), basement membrane inhibition of keratinocyte differentiation (88), transactivation of the cytotactin/tenascin and neural cell adhesion molecule (N-CAM) promoters by specific homeodomain proteins (128), and modulation of N-CAM in dissociated versus aggregated p19 embryonal cells (170). How the ECM influences adipocyte differentiation is yet to be addressed in appropriate molecular detail.

The ECM of adipose tissue interconnects adipocytes and gives rise to fat cell clusters in vitro and to fat lobules of adipose tissue in vivo. During adipocyte differentiation, drastic changes occur in cell morphology, cytoskeletal components, and the level and type of ECM components secreted as described in section VC. An early ultrastructural change seen in in vivo adipocyte differentiation is the deposition of collagen at the cell-ECM border and extracellular basement membrane biogenesis (183). Fibroblasts in developing fat tissue have loose interactions with the ECM that may be necessary for the morphological changes that accompany differentiation or to keep cells in close proximity and thereby increase their exposure to juxtacrine differentiation signals. Electron microscopy reveals that preadipose 3T3-F442A cells have limited granular ECM deposits. In contrast, 3T3-F442A adipocytes appear interconnected by abundant ECM rods and fibers and resemble the fat cell clusters seen in lobules of adipose tissue (146). These changes may be related to the loose interaction of the basement membrane with adipose cells that has been observed in fat tissue. Many of the ECM components, for example, laminin and entactin/nidogen, are known to interact with each other and the cell surface. Modulation of ECM components could permit release of cell-cell adhesion and remodeling of cell components. These changes might be necessary for cellular reorganization and could provide a permissive or instructive environment for the expression of adipocyte genes.

Subjecting preadipocytes to differentiation conditions in the presence of ECM components demonstrates their influence on adipocyte differentiation. Type I and III collagen, fibronectin, and poly-L-lysine, as well as -integrins are negatively correlated with the differentiation of preadipocyte cell lines (11, 24, 221, 257, 290), whereas increases in type IV collagen, entactin, and an unorthodox laminin complex accompany the adipocyte differentiation process (13, 195). Culture of 3T3-F442A preadipocytes on fibronectin matrices decreases gene expression of lipogenic enzymes and leads to decreased triglyceride accumulation (257), possibly by interfering with the cytoskeletal and morphological changes necessary for adipocyte differentiation. The inhibitory effect of fibronectin is not observed by its addition to culture media, and this inhibition is overcome by keeping cells in a rounded configuration. These observations indicate that the inhibitory effect of fibronectin requires cell spreading. Cytochalasin D, an agent that disrupts actin filaments, overcomes the inhibitory effects of fibronectin (257), further indicating that cytoskeletal rearrangement is a prerequisite for terminal differentiation. In a separate study, differentiation of 3T3-F442A cells has been shown to be accelerated by cytochalasin D treatment, perhaps by allowing earlier cytoskeletal remodeling. Furthermore, with long-term cytochalasin D treatment, a small proportion of differentiation-defective 3T3-C2 cells undergo morphological and biochemical conversion to adipocytes (197). Addition of soluble fibronectin and growth on fibronectin matrices markedly decreases differentiation of ST-13 preadipocytes. This is reversed by an antibody against the ₅ ,₁-integrin and by a short peptide corresponding to the cell attachment domain of fibronectin. Interestingly, differentiation of these cells, on the other hand, is stimulated by the addition of a thermolysin digest of fibronectin (76). The adipocyte-stimulating activity is in the NH₂-terminal fibrin binding domain and can be activated by use of specific matrix metalloproteinases (76, 77). These experiments suggest that fibronectin has the ability to both inhibit and stimulate adipocyte differentiation, depending on the state of the molecule.

As described in section VC, changes in expression of specific types of collagen accompany adipocyte differentiation. Use of ethyl-3,4-dihydroxybenzoate (EDHB), a specific inhibitor of active collagen synthesis, highlights the role of collagen in modulating adipocyte differentiation. Exposure of TA1 cells to EDHB preceding confluence and during the initial stage of differentiation prevents cells from entering the terminal differentiation program; this demonstrates that differentiation of preadipose cells into adipocytes requires active synthesis of collagen (124). A role for proteoglycans in adipocyte differentiation is suggested by the observation that proteoglycan metabolism is altered during 3T3-L1 preadipocyte differentiation (29, 182). Levels of versican/CSGP-I, a chondroitin 4-sulfate proteoglycan, are increased early during 3T3-L1 cell differentiation. This increase appears to correlate with the increased viscosity of the culture medium that occurs during differentiation. The role of ECM in the differentiation process is confirmed in primary preadipocyte studies (108, 214, 215, 279). The source or type of ECM dictates the influence of ECM substrata on primary porcine preadipocyte differentiation (108). Laminin, in particular, may play a critical role in morphological changes of porcine preadipocyte development. All of the above studies clearly indicate that ECM components modulate adipocyte differentiation, perhaps by release of cell-cell adhesion, thereby permitting changes in cell morphology or volume (29). It remains to be determined whether ECM components could be directly involved in initiating or transducing signals for adipose differentiation or merely allow cells differential access to the growth and/or inhibitory factors present in the surrounding environment.

	VII. TRANSCRIPTION FACTORS CRITICAL FOR ADIPOCYTE DIFFERENTIATION

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Adipocyte differentiation involves communication of extracellular signals and those of the ECM environment to the nucleus. This leads to a coordinate regulation of adipocyte-specific gene expression resulting in the mature adipocyte that is highly specialized for energy storage and homeostasis. As presented above, many classes of molecules transduce inductive and inhibitory signals from the environment. Although the full complement of proteins involved in this process remains to be determined, ultimately the PPAR and C/EBP family of transcription factors must function cooperatively to transactivate adipocyte genes and thereby bring about adipocyte differentiation. The role of transcription factors of the PPAR and C/EBP families in adipocyte differentiation has been extensively reviewed recently (27, 153, 167), and we present an overview only.

The PPARs belong to type II nuclear hormone receptor family and form heterodimers with the RXR (131, 231). The PPARs are activated by a variety of structurally dissimilar compounds, including the thiazolidinedione class of antidiabetic drugs, Wy-14, 463, and clofibrate. The PPARs regulate transcription through binding of PPAR-RXR heterodimers to a response element consisting of a direct repeat of the nuclear receptor hexameric DNA recognition motif (PuGGTCA) spaced by one nucleotide (DR-1) (271). Peroxisome proliferator-activated receptor- is the most adipose specific of the PPARs, and it is induced before transcriptional activation of most adipocyte genes. Low but detectable expression of PPAR- also occurs in liver and hematopoietic cells (25, 62). The expression of PPAR- has been shown to be sufficient to induce growth arrest as well as to initiate adipogenesis in exponentially growing fibroblast cell lines, demonstrating its critical role in the regulation of adipocyte differentiation (6, 120, 272). Moreover, retroviral-mediated ectopic expression of PPAR- in the presence of PPAR activators decreases myoblast-specific gene expression and directs these cells into the adipocyte lineage (120). Both synthetic and natural ligands of PPAR- have recently been identified. Thiazolidinediones, a new class of drugs which increase insulin sensitivity, can directly bind and activate PPAR- and have been shown to stimulate adipose conversion (140, 155, 227). Thiazolidinediones are strongly adipogenic for fibroblasts and myoblasts, especially for those expressing PPAR-, with 15-deoxy-D-PGJ₂ , the most potent activator (72, 141). Whether 15-deoxy-D-PGJ₂ is the major endogenous ligand for PPAR- is not yet known (27). The observations reporting that PPAR- is highly expressed in adipocytes and that its ectopic expression is capable of triggering the entire program of adipogenesis in fibroblast and muscle cells has led to the hypothesis that PPAR- can regulate development of the adipocyte lineage (272). Although PPAR- plays a critical role in the induction and maintenance of the fully differentiated adipocyte phenotype, its participation in the adipocyte lineage determination process remains unclear. Retinoic acid but not potent PPAR activators can commit ES cells into the adipocyte lineage, suggesting that RAR may be involved in the initial steps of adipose cell development (52).

The role of other PPAR isoforms in adipogenesis remains to be resolved. Peroxisome proliferator-activated receptor- was also reported to be able to induce significant adipocyte differentiation in response to strong PPAR activators. Because this isoform is only weakly expressed in adipocytes and has been reported to be less adipogenic than PPAR-, its precise role in triggering and/or maintaining the adipocyte phenotype remains unclear (28). Although PPAR- is not adipocyte specific, this PPAR isoform is highly expressed in adipose tissue. It is upregulated very early during adipocyte differentiation and has been suggested to play an important role in adipogenesis (7). Involvement of PPAR- in the trans-differentiation of myoblasts to adipoblasts triggered by either fatty acids or thiazolidinediones has been recently postulated by Grimaldi et al. (100). However, a role for PPAR- in triggering trans-differentiation seems very unlikely given that the level of PPAR- is high in myoblasts and is only marginally increased with thiazolidinedione treatment (266). Moreover, transcriptional activation of PPAR isoforms by various PPAR activators demonstrates that thiazolidinediones are potent activators of PPAR- but not PPAR- (28). Transfection of 3T3-C2 cells with a PPAR- expression construct has been reported to confer fatty acid inducibility on the aP2 gene and the FAT gene (7). However, retroviral expression and activation by bromopalmitate of PPAR- in NIH 3T3 cells does not stimulate adipogenesis (28). Although a partial role of PPAR- in mediating the transcriptional effects of fatty acids on preadipocyte differentiation cannot be ruled out, its role in adipose tissue development still remains to be demonstrated.

Members of the C/EBP family were the first transcription factors demonstrated to play a major role in adipocyte differentiation. These transcription factors have a basic transcriptional activation domain and an adjoining leucine zipper motif, which provides the ability for homo- and heterodimerization. Isoforms of C/EBP are expressed in tissues, such as liver, that metabolize lipid and cholesterol-related compounds at high rates (43). Studies of adipocyte differentiation, primarily in 3T3-L1 cells, provide extensive evidence for C/EPB- function in adipocyte differentiation (27, 153, 166, 167). Although not strictly adipocyte specific, C/EPB- is expressed just before the transcription of most adipocyte-specific genes is initiated. CCAAT/enhancer binding protein- binds and transactivates the promoters of several adipocyte genes, including aP2, SCD1, GLUT-4, PEPCK, leptin, and the insulin receptor. Mutation of the C/EPB- site in these genes abolishes transactivation (121, 132, 172, 223). In some instances, constitutive expression of C/EPB- is sufficient to induce differentiation of 3T3-L1 cells in the absence of hormonal agents, and expression of antisense C/EPB- mRNA in 3T3-L1 preadipocytes prevents differentiation (158, 159). Moreover, C/EPB- can efficiently promote adipogenesis in a variety of mouse fibroblastic cells, including those that have little or no spontaneous capacity to develop into adipocytes (75). In view of its known antimitotic activity, C/EPB- has also been indicated to function in the termination of the mitotic clonal expansion that occurs early in the differentiation program and seems to be involved in the maintenance of the adipocyte phenotype through autoactivation of the C/EBP- gene (42). Together, these findings provide evidence that C/EPB- is both required and sufficient to induce adipocyte differentiation. Generation of C/EPB- knockout mice supports the physiological role of C/EPB- in adipose tissue development, since C/EBP- null mice fail to develop WAT (289). However, this study probably addresses the role of C/EPB- in adipocyte maturation rather than its potential function in the initiation and/or commitment of mesenchymal precursors. Although mature white adipocytes are absent in C/EPB- (/) mice, the presence of committed preadipocytes already expressing early markers of differentiation has not been assessed, and their existence cannot be excluded. Fine-tuning of the control of adipocyte gene expression by C/EBP proteins involves homo- and heterodimerization between the C/EBP-, C/EBP-, and C/EBP- isoforms. Each isoform has a distinct temporal and spatial expression pattern during adipocyte differentiation (32). Whereas C/EBP- expression occurs relatively late in differentiation, the - and -isoforms of C/EBP are present in preadipocytes, and their levels increase transiently early in differentiation. By late differentiation, C/EBP- decreases to 50% of its initial level, and C/EBP- is nearly undetectable (32). Neither C/EBP-, -, nor - is adipocyte specific (20, 32), although C/EBP- is expressed at high levels in the mature adipocyte and adipose tissue (32, 306). CCAAT/enhancer binding protein- and possibly C/EBP- seem to function early as transcriptional activators in the sequence of events leading to adipocyte differentiation. Conditions that cause expression of either the - or -isoforms accelerate adipogenesis in 3T3-L1 preadipocytes (167). Ectopic expression of C/EBP-, particularly the liver-enriched transcriptional activator protein spliced form, activates the expression of PPAR- in NIH 3T3 fibroblasts exposed to dexamethasone, insulin, and fetal bovine serum and stimulates their conversion into committed preadipocytes (300). Expression of PPAR- is induced by coexpression of C/EBP- and C/EBP-. Although this suggests that these two C/EBP isoforms may heterodimerize to directly regulate PPAR- (299), the increased PPAR- expression may also be the indirect effect of onset of adipocyte differentiation resulting from C/EBP- action. Taken together, current data suggest that an increase in C/EBP- above a threshold level induces expression of PPAR-. Upon ligand activation, PPAR-, in concert with C/EBP-, leads to the full adipocyte differentiation program.

The observation that in 3T3-L1 preadipocytes C/EBP- and C/EBP- levels are increased by MIX and dexamethasone, respectively (32, 306), has led to the suggestion that C/EBP- and C/EBP- might relay the adipogenic effects of these agents early in adipocyte differentiation. Expression of ectopic C/EBP- in NIH 3T3 cells obviates the MIX requirement for their differentiation, indicating that C/EBP- may be a primary effector of MIX action in adipocyte differentiation. The observed induction of C/EBP- by dexamethasone could lead to the formation of C/EBP--C/EBP- heterodimers, and these were postulated to be more transcriptionally active than C/EBP- homodimers. However, examination during the initial stages of differentiation of dexamethasone-treated NIH 3T3 fibroblasts that ectopically express C/EBP- indicates that C/EBP- only minimally contributes to functional C/EBP complexes (299). Moreover, dexamethasone remains an absolute requirement for adipocyte differentiation of NIH 3T3 fibroblasts ectopically expressing high levels of both C/EBP- and C/EBP-, indicating that C/EBP- cannot substitute for the role of dexamethasone in the differentiation process (299).

C/EBP homologous protein-10 (CHOP-10) is an another member of the C/EBP family. It displays overall sequence similarity to C/EBP proteins in the DNA binding and dimerization domain. However, the basic region of CHOP-10 deviates considerably in sequence from that of other C/EBP proteins, and CHOP-10-C/EBP heterodimers are unable to bind to a common class of C/EBP sites. With respect to such sites, CHOP-10 appears to function as a negative modulator of the activity of C/EBP transcription factors (275). CHOP-10 is absent under normal conditions and is induced by cellular stresses; its function under normal circumstances is still unclear.

A potential role of bHLH in activating the adipocyte differentiation program has recently been suggested by the observation that overexpression of Id3 prevents adipocyte differentiation (177). Ids are a group of ubiquitous nuclear proteins that possess the helix-loop-helix domain but are missing the basic DNA binding domain. Ids are believed to block muscle differentiation by competing with myogenic bHLH proteins for dimerization with their bHLH binding partner, E12/E47, which binds to an E-box motif to transactivate muscle-specific genes (126). In 3T3-F442A cells as well as in primary preadipocyte cultures, downregulation of Id2 and Id3 has been shown to precede adipocyte differentiation. A fall in Id3 seems to be a specific event of the adipocyte differentiation program; its mRNA declines very early after confluence and is not stimulated when fresh serum is added to confluent 3T3-F442A cells. On the other hand, Id2 and Id3 mRNA levels are not reduced in the differentiation-defective 3T3-C2 cells (177). These results indicate that Id3 may be a negative regulator of fat cell formation by preventing an as yet unidentified bHLH protein from activating the adipocyte differentiation program. To date, there is no candidate for such a protein. SREBP-1c/ADD1 is a bHLH protein that is expressed abundantly in adipose tissue (136) but is considered not capable of heterodimerization with Ids (177); SREBP-1c/ADD1 displays a dual DNA specificity, binding to both an E-box motif and non-E-box motif (SRE-1) (137). A role in adipocyte differentiation is suggested by the early increase of its mRNA levels during adipocyte differentiation and by its transactivation of several key lipogenic genes (64, 136). SREBP-1c/ADD1 has been shown to increase fatty acid and fat synthesis, and this has been attributed in part to its proposed influence on PPAR- activity; it has been hypothesized to be involved in the generation of endogenous ligands for PPAR- (27). Expression of SREBP-1c/ADD1 is also increased during osteoblast differentiation, where it is correlated with expression of an osteoblastic phenotype-related gene, indicating that increased expression of this protein is not specific to adipocyte differentiation. However, this observation appears to further illustrate the close relationship between the osteoblast and adipoblast lineages (228).

Another bHLH protein, twist, discussed in section III, is expressed at high levels in WAT and brown adipose tissue, primarily in stromal cells. A decrease in twist expression is observed upon differentiation of 3T3-L1 cells and primary preadipocytes. Like Ids, twist is not adipocyte specific; a similar pattern of expression occurs in osteogenic cell lines (85). Twist, when ectopically expressed, inhibits myoblast differentiation by preventing transactivation of muscle target genes by MEF2. In addition, twist, like Ids, dimerizes with E proteins, thereby blocking binding of myogenic bHLH transcription factor to DNA (222, 254). Although modulation of twist expression during adipocyte differentiation strengthens the potential role of this bHLH transcription factor in adipose tissue development, its precise role in this process remains difficult to predict at this point.

Intensive efforts dedicated to the isolation of regulatory genes for adipocyte differentiation have resulted in the identification of the C/EBP and PPAR family of transcription factors, which play a central role in the terminal differentiation of adipocytes. In addition, the expression pattern of the above-described helix-loop-helix transcription factors in the adipocyte lineage is consistent with the concept that other transcription factors may function in, or modulate, different stages in adipocyte development. Use of new cell culture models such as ES cells, recently shown to differentiate at high efficiency into adipocytes in certain culture conditions (52), may provide a system for the identification of new adipogenic regulatory genes or those that function at earlier stages.

	VIII. CONCLUSIONS

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The elucidation of the cooperative function of PPAR- and C/EBP- as the key transcription factors for adipocyte gene activation and differentiation has been pivotal to our understanding of adipogenesis at the molecular level. A variety of agents influence adipocyte differentiation, although their effects are somewhat dependent on the preadipose cell culture models and culture conditions employed. With this variability in mind, a general picture emerges, suggesting that the multitude of diverse regulatory signals likely converges at a few classic signal transduction pathways. We can just now begin to connect the dots from hormonal and growth factor signals to specific nuclear events. It is likely a detailed picture will emerge over the next several years. The inhibitory actions of pref-1, a member of the EGF-repeat family, suggest that new classes of important regulatory molecules remain to be discovered. Less is known about the signaling processes subsequent to cell-cell or cell-matrix interactions that modulate transcriptional activity and adipocyte differentiation, and this area warrants future detailed examination. Understanding how the various molecules regulate adiposity and energy balance in physiological and pathophysiological situations remains an important area for future research and may lead to the development of novel therapeutic approaches to human obesity.

	ACKNOWLEDGEMENTS

  We thank D. Kachinskas for valuable help in preparation of the manuscript.

	FOOTNOTES

   The work from the authors' laboratory was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-50828 (to H. S. Sul).

   Present address of F. M. Gregoire: Rowe Genetics Program, Dept. of Pediatrics, Univ. of California, Davis, CA 95616.

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