WAT 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. 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. The committed preadipocyte maintains the capacity for growth but has to with draw from the cell cycle before adipose conversion.
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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).
3.1. Growth Arrest
In preadipose cell lines as well as in primary preadipocytes, growth arrest and not cell confluence or cell-cell contact appears to be required for adipocyte differentiation. Although confluence 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).
C/EBP-Î± and PPAR-Î³ transcription factors have been shown to transactivate adipocyte specific genes. Both C/EBP-Î± and PPAR-Î³ also appear to be involved in the growth arrest that is required for adipocyte differentiation. McKnight and coworkers (276) have demonstrated the antimitotic activity of C/EBP-Î± through the use of a C/EBP-Î±-estrogen receptor fusion protein. Activation by estrogen treatment results in cessation of cell growth as assessed by cell number and DNA synthesis (276). In PPAR-Î³-expressing cells, cell cycle withdrawal is accompanied by a decrease in the DNA binding. Therefore, C/EBP-Î± and PPAR-Î³ may act cooperatively to bring growth arrest (6). Although C/EBP-Î± and PPAR-Î³ 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.
3.2. Clonal Expansion
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). 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.
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 post confluent preadipocytes, whereas gas1and 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.
3.3. Early Changes in Gene Expression
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). LPL 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.
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The early expression of C/EBP and PPAR is logical given their subsequent involvement in terminal differentiation by transactivation of adipocyte-specific genes. PPAR-Î³ is largely adipocyte specific and is expressed at low but detectable levels in preadipocytes. Its expression rapidly increases after hormonal induction of differentiation. A transient increase in the expression of C/EBP-Î² and C/EBP-Î´ isoforms precedes the increase in PPAR-Î³ expression (27, 167, 299). The subsequent decrease of C/EBP-Î² and C/EBP-Î´ in early to intermediary stages of differentiation is concomitant with the induction of C/EBP-Î± mRNA. This increase in C/EBP-Î± expression occurs slightly before the expression of adipocyte-specific genes (27, 153, 167).
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. 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.
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%, while secretion of type IV collagen and entactin/nidogen increases (13, 290).
The amount of pericellular fibronectin, as well as cellular synthesis of fibronectin, decreases by four- to five folds during differentiation (10).
Preadipocyte factor-1 (pref-1), a preadipocyte protein with epidermal growth factor (EGF)-like repeats, 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
3.4. Late Events and Terminal 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-phosphatedehydrogenase 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 Î²1-adrenergic receptors and an increase in the Î²2- and the Î²3-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, such as: aP2 and perilipin, a lipid droplet-associated protein (94).
Adipocytes produce a number of secreted products, including: monobutyrin, an angiogenic agent; adipsin; 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).
3.5. Factors that modulate adipocyte diferentiation
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. ECM proteins may play an important role in modulating adipocyte differentiation by permitting the morphological changes and adipocyte specific gene expression that accompany 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.
3.5.1. Growth hormone and IGF-I
Studies addressing the role of growth hormone (GH) and IGF-I in adipocyte differentiation illustrate potential problems in comparing results obtained with different cell culture models under various culture conditions. A role for GH in adipocyte differentiation was first reported by Green et al. (93). Growth hormone promotes differentiation and sensitizes the cells to the mitogenic effects of IGF-I for clonal expansion (46, 93, 102). Sonenberg and co-workers (46, 102) suggested that GH may promote adipose conversion by inducing an antimitogenic state that is accompanied by decreased synthesis of the ECM proteins fibronectin and collagen. Growth hormone stimulates IGF-I gene transcription. In contrast to observations in preadipose cell lines, no stimulatory effect of GH is observed in primary preadipocyte cultures.
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A requirement of IGF-I or pharmacological concentrations of insulin in adipocyte differentiation has been clearly demonstrated. Insulin-like growth factor-I also stimulates adipogenesis of primary rat, rabbit, and porcine preadipocytes (57, 190, 211), indicating that this growth factor may be an essential regulator of fat cell formation.
Clonal and primary preadipocytes also secrete insulin-like growth factor binding proteins (IGFBPs) in a differentiation-dependent manner, indicating that IGFBPs may be important in modulating IGF-I action in adipogenesis (22, 40, 190, 283). The mechanisms of action of IGF-I/IGFBPs are not well understood, but they are most likely acting in an autocrine/paracrine manner. The adipogenic effects of IGF-I indicate the involvement of a phosphorylation-dephosphorylation mechanism, subsequent to IGF-I receptor tyrosine phosphorylation, in intracellular signaling during adipocyte differentiation.
Recently, a serine/threonine kinase Akt (PKB) also has been demonstrated to be involved in adipocyte differentiation. Akt is activated by insulin and certain growth factors, and evidence indicates it functions as a downstream effector of phosphatidylinositol 3-kinase pathway. Expression of constitutively active Akt in 3T3-L1 cells results in their spontaneous differentiation into adipocytes in the absence of the normal inducing agents, suggesting the involvement of Akt-mediating signaling in adipocyte differentiation (142).
3.5.2. 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 their mitogenic effects, since cell growth and differentiation are usually mutually exclusive. As previously discussed, 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-Î±, in adipose tissue development.
Epidermal growth factor inhibits differentiation of mouse, rat, and human preadipocytes (105, 233, 282), and subcutaneous administration of EGF to newborn rats results in a substantial decrease in fat pad weight, which suggests a delayed formation of adipocytes from preadipocytes (238). However, EGF is not always inhibitory. Differentiation of 3T3-L1 preadipocytes grown in serum-free medium has been reported to depend on EGF or platelet-derived growth factor (PDGF) (230). Chronic treatment of porcine preadipocyte cultures with EGF does not significantly alter their differentiation.
The role of basic fibroblast growth factor (bFGF) and PDGF in adipocyte differentiation is not clear. Basic fibroblast growth factor has been shown to have antiadipogenic effects in several preadipocyte cell lines under serum-containing conditions (109, 184, 185), whereas it has no effect in serum-free conditions (230). Moreover, in serum-free conditions, exposure of human preadipocytes to varying concentrations of bFGF has no effect on the number and morphology of differentiating cells (105) and either a modest or no stimulatory effect on rat preadipocytes (236, 282).
In most cell culture models, TGF-Î² is a potent inhibitor of adipocyte differentiation (160, 203, 237, 253, 282). A possible mechanism for TGF-Î² inhibition of adipocyte differentiation may be via increasing synthesis of ECM components.
Inhibition of in vitro adipocyte differentiation, assessed by triglyceride accumulation and expression of various marker mRNAs, is also reported for a number of cytokines. Interleukin-11 has a dose-dependent inhibitory effect. Inhibition is dominant over the effect of standard inducing agents (193, 194). Interferon-Î³ and interleukin-1Î² inhibit the adipoconversion (95, 134, 199).
Tumor necrosis factor-Î± decreases LPL synthesis and inhibits adipocyte differentiation. TNF-Î±-mediated reversal of adipocyte differentiation has been shown to be associated with the downregulation of C/EBP-Î± and induction of c-myc expression (189, 259, 297). In addition, TNF-Î± treatment causes a rapid decrease in the levels of PPAR-Î³ mRNA and protein, as well as a parallel decrease in PPAR-Î³ DNA binding activity that precedes the decrease in C/EBP-Î± and aP2. This suggests that the downregulation of PPAR-Î³ may be a mechanism whereby TNF-Î± exerts its effects in the mature adipocyte (301, 311).
Furthermore, it has been shown that PPAR-Î³ is a phosphoprotein that undergoes EGF-stimulated MEK and MAPK-dependent phosphorylation. This phosphorylated form is less active in transactivation of adipocyte genes and in promoting adipogenesis (1, 30, 118). This therefore suggests that some of the growth factors inhibitory to adipocyte differentiation might act through the MAPK pathway to phosphorylate PPAR-Î³. However, insulin treatment, which is known to increase lipid accumulation during adipocyte differentiation, also results in PPAR-Î³ phosphorylation (311).
3.5.3. Nuclear hormone superfamily
Members of the nuclear hormone superfamily, including glucocorticoids, 3,3',5-triiodothyronine (T3), and RA, influence adipocyte differentiation. Their action in adipocyte differentiation is not well characterized at the molecular level, but these hormones, in general, exert nuclear effects by binding to their respective intracellular hormone receptors.
Depending on the origin of the cells and culture conditions, glucocorticoid treatment is either required for differentiation or acts to only accelerate this process. It has been demonstrated that glucocorticoids induce expression of C/EBP-Î´. This increase may contribute to the formation of C/EBP-Î´-C/EBP-Î² heterodimers, which in turn may lead to PPAR-Î³ expression (299). Glucocorticoid effects have been shown to be mediated through increased metabolism of arachidonic acid leading to an increase in production of prostacyclin, which in turn increases intracellular cAMP (4).
The ability of RA to affect various differentiation processes including the terminal events of the adipocyte differentiation program has been recognized for several years. When used at supraphysiological concentration, RA inhibits adipocyte differentiation of preadipocyte cell lines and primary porcine preadipocytes (60, 263). Retinoic acid addition either before or after treatment with inducing agents does not affect differentiation, indicating that RA acts at an early stage in differentiation. This finding is supported by the observation that RA treatment prevents induction of C/ EBP-Î± and interferes with the mechanisms that induce as well as maintain PPAR-Î³ expression. These actions of RA seem to be predominantly mediated by liganded RA receptors (RARs) rather than retinoid X receptors (RXRs) (38, 302). Moreover, recent evidence indicates that the inhibitory effects of RA occur before PPAR-Î³ expression by blocking C/EBP-Î² induction (232).
3,3*,5-Triiodothyronine also has been implicated in the terminal differentiation of Ob17 preadipocytes (82). The role of T3 appears to be restricted to the Ob17 preadipose cell line, since no clear requirement for T3 is observed in other preadipocyte culture models, including the 3T3-L1 and rat, porcine, or human primary preadipocyte cultures (57, 103, 104, 230, 236, 264, 293).
Mature adipocytes and cultured preadipocytes produce significant amounts of prostaglandins (PGs), including PGF2a, PGE2, PGD2, and PGI2 (122, 215). Prostaglandin E2 is a strong antilipolytic compound, and PGF2a and PGI2 have been shown to modulate preadipocyte differentiation. Prostaglandin D2 may be endogenous ligand for PPAR-Î³ and therefore acts as adipogenic signal (141).
PGF2Î± stimulates mRNA expression and production of TGF-Î±, both in undifferentiated and differentiated cells. Both PGF2Î± and TGF-Î±, which are inhibitors of adipocyte differentiation, are produced locally in adipose tissue. Therefore, stimulation of TGF-Î± expression by PGF2Î± could represent an amplification mechanism to modulate preadipocyte differentiation and adipocyte function within adipose tissue (156).
In contrast to the inhibitory role of PGF2Î±, a potent and specific adipogenic role has been attributed to prostacyclin (PGI2), one of the major metabolites of arachidonic acid. Carbacyclin, a stable prostacyclin analog, has been shown to act by means of two intracellular signaling pathways known to synergize in inducing adipocyte differentiation, i.e., concomitant elevation of cAMP and free intra cellular calcium (280, 281). Prostacyclin has also been reported to be an activator of the three known mammalian PPARs (Î±, Î´, and Î³) and to be the most effective activator for PPAR-Î´ described to date. The paracrine adipogenic effect of PGI2 has also been reported to be controlled by angiotensin II. In vivo, this paracrine mode of action may represent a crucial biological signal in the hyperplastic development of adipose tissue known to occur once adipose cells reach their maximal size.
3.5.5. cAMP, G proteins, and protein kinase C
The G proteins GsÎ± and GiÎ± have been shown to mediate adipocyte differentiation in 3T3-L1 cells independent of adenylyl cyclase (87, 288). Expression of the inhibitory subunit GÎ±i-2 promotes lipid accumulation and agents that activate GsÎ± block differentiation. These effects of GsÎ± and GÎ±i-2 are exerted at ambient or elevated intracellular cAMP levels, demonstrating that this critical role of G proteins in adipocyte differentiation is independent of adenylyl cyclase (288).
Although total protein kinase C (PKC) activity is reduced during differentiation, not all isoforms have lowered expression. PKCÎµ expression occurs only during differentiation. PKC-Î± and PKC-Î¶ decrease during rat preadipocyte differentiation suggesting a possible involvement of PKC-Î¶ in the postreceptor signaling pathway of insulin (149). Ectopic expression of PKC-Î· is also reported to alter the expression of cyclins and CDK inhibitors and induce adipogenesis in NIH 3T3 fibroblasts (161). These findings indicate that the PKC pathway of signal transduction is part of a highly complex system that likely exerts negative as well as positive effects on the adipocyte differentiation process.
IBMX accelerates the differentiation of preadipose cell lines and primary preadipocytes. It has been shown to increase expression of C/EBP-Î², required for subsequent PPAR-Î³ expression and adipocyte differentiation. IBMX 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 Gi, increasing cAMP levels.
3.5.6. Pref-1, Inhibitory action in adipogenesis
Pref-1 is an 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). The role of EGF repeats in other molecules leads to several hypotheses for pref-1 action in adipocyte differentiation. The EGF repeat unit is a 35- to 40-amino acid sequence characterized by highly conserved spacing of six cysteine residues that form three disulfide bonds. Members of the EGF-repeat family of proteins are membrane-bound or secreted proteins that act on cell growth and differentiation in an astonishing array of biological settings (12, 154, 192). A single EGF-like domain is the functional unit of EGF, TGF-Î±, and other growth factors that interact with the EGF receptor (33). The EGF-repeat family also includes proteins of the blood-clotting cascade, the LDL receptor, and several multidomain ECM proteins and cell adhesion molecules with a demonstrated role in cell guidance and development.
Although the mechanism of pref-1 inhibition of adipocyte differentiation remains to be determined, given the importance of cell shape modulation and the ECM environment in adipocyte differentiation, transmembrane pref-1 may possibly function by the interaction of its EGF like domains with EGF-like or other protein domains present in ECM molecules, thereby maintaining the preadipose phenotype. These could include versican and laminin. Both contain EGF-repeat domains and are modulated during adipocyte differentiation.
3.5.7. Extracellular matrix components
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. 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). Many of the ECM components are known to interact with each other and the cell surface. Modulation of ECM could allow the release of cell-cell adhesion and remodeling of cell components. These changes might be necessary for cellular reorganization and could provide a permissive environment for the expression of adipocyte genes.
Type I and III collagen, fibronectin, and poly-L-lysine, as well as Î²-integrins are negatively correlated with the differentiation of preadipocyte cell lines (11, 24, 221, 257, 290), whereas increases in type IV collagen, entactin, and laminin accompany the adipocyte differentiation process (13, 195).
3.6. Transcription factors
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.
3.6.1. PPAR Family
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. 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 (PuGG- TCA) spaced by one nucleotide (DR-1) (271). Peroxisome proliferator-activated receptor-Î³ 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-Î³ also occurs in liver and hematopoietic cells (25, 62). The expression of PPAR-Î³ 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).
3.6.2. C/EBP Family
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). Although not strictly adipocyte specific, C/EPB-Î± is expressed just before the transcription of most adipocyte-specific genes is initiated. CCAAT/enhancer binding protein-Î± 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-Î± site in these genes abolishes transactivation (121, 132, 172, 223). C/EPB-Î± is both required and sufficient to induce adipocyte differentiation.
The control of adipocyte gene expression by C/EBP proteins involves homo- and heterodimerization between the C/EBP-Î±, C/EBP-Î², and C/EBP-Î´ isoforms. Each isoform has a distinct temporal and spatial expression pattern during adipocyte differentiation (32). Whereas C/EBP-Î± expression occurs relatively late in differentiation, the Î²- and Î´-isoforms of C/EBP are present in preadipocytes, and their levels increase transiently early in differentiation. By late differentiation, C/EBP-Î² decreases to 50% of its initial level, and C/EBP-Î´ is nearly undetectable (32). Neither C/EBP-Î±, -Î², nor -Î´ is adipocyte specific (20, 32), although C/EBP-Î± is expressed at high levels in the mature adipocyte and adipose tissue (32, 306). CCAAT/enhancer binding protein-Î² and possibly C/EBP-Î´ seem to function early as transcriptional activators in the sequence of events leading to adipocyte differentiation. Expression of PPAR-Î³ is induced by coexpression of C/EBP-Î² and C/EBP-Î´. Although this suggests that these two C/EBP isoforms may heterodimerize to directly regulate PPAR-Î³ (299), the increased PPAR-Î³ expression may also be the indirect effect of onset of adipocyte differentiation resulting from C/EBP-Î² action. Taken together, current data suggest that an increase in C/EBP-Î² above a threshold level induces expression of PPAR-Î³. Upon ligand activation, PPAR-Î³, in concert with C/EBP-Î±, leads to the full adipocyte differentiation program.
4. Adipose tissue engineering
Regenerative medicine is a multidisciplinary field of research which involves the use of biomaterials, growth factors, and stem cells to repair, replace, or regenerate tissues and organs damaged by injury or disease (Guilak, 2002; Sundelacruz and Kaplan, 2009) and has definitely evolved in parallel with advances in the biotechnological field.
Tissue engineering enhances the tissues regeneration through the use of biodegradable scaffolds combined with in vitro cultured cells (Atala, 2006; Vacanti and Langer, 1999; Yen and Sharpe, 2006; Zuk, 2008). Regarding the cellular component, stem cells are ideal candidates for regenerative medicine due to their ability to self-renew and to commit to multiple cell lineages (Gimble et al., 2007; Pittenger et al., 1999).
Stem cells for regenerative medicine applications should meet the following criteria (Gimble et al., 2007): i) abundance of cells (millions to billions); ii) minimally invasive procedure with minimal morbidity harvest; iii) differentiation potential along multiple cell lineages in a controllable and reproducible manner; iv) safe transplantation to either an autologous or allogeneic host; v) possibility of isolation in accordance with the current Good Manufacturing Practice guidelines
Several sources of stem cells likely fulfill the criteria but human adipose derived stem cells (hADSCs) have multiple advantages (Fraser et al., 2006); NU with the increased occurrence of obesity, subcutaneous adipose tissue is accessible and thus hADSCs can be harvested in large quantities with minimal risk. In addition, adipose tissue yields manifold greater numbers of mesenchymal stem cells (MSCs) compared to bone marrow (Fraser et al., 2006). Due to their secretory profile, hADSCs delivered into injured or diseased tissue, stimulate the recovery in a paracrine manner. These cells were shown to modulate the "stem cell niche" of the host by stimulating the recruitment of endogenous stem cells to the site of injury and promote their differentiation along the required lineage pathway . In addition, hADSCs secrete nearly all of the growth factors that take part in normal wound healing [10-13]. Since 2001, when the existence of stem cells within this tissue was reported (Zuk et al., 2001), adipose tissue has acquired increased importance as a stem cell source for a wide range of potential applications in regenerative medicine strategies.
Resection of tumors as well as trauma or congenital abnormalities often result in contour defects due to loss of soft tissue, largely composed of subcutaneous adipose tissue. Besides the reconstruction of the functional tissue, a modern requirement in regenerative medicine is also the aesthetic restoration of the resulting imperfections . Classical clinical strategies for ATE include the use of autologous fat implants, which are considered to be the ideal filling material in terms of biocompatibility, immune response and avoidance of graft rejection . However, adipose tissue transplantation yields unpredictable results, due to varying degrees of graft resorption over time (40%-60% volume loss) and lack of sufficient revascularization [3,4].
The alternative use of synthetic surrogates (Teflon, silicone implants) or allogenic materials, like bovine collagen, have the advantage of endless supply, but clinical experiences revealed various deficiencies, such as rupture, capsular contracture, dislocation, suboptimal biocompatibility of the implants and allergic reactions [5,6].
Modern strategies in current ATE applications involve the design of 3D cell-scaffold bioconstructs obtained by preseeding the scaffold with undifferentiated cells. In order to achieve in situ functional de novo tissue, the embedded hADSCs are committed towards the adipogenic lineage by subjecting the bioconstructs to in vitro adipogenic conditions. Subsequently, the engineered tissue is expected to be structurally, mechanically and functionally integrated to the implantation site. Overall, the most important feature of this modern strategy is the achievement of a long-term and predictable clinical application result ensured by the control of the scaffold's composition, implanted cell number and the differentiation status and kinetics. After implantation, hADSCs remain viable at the wound site and secrete growth factors in a continuous and regulated manner in response to environmental cues, just as it occurs in the natural wound healing process . Consequently, at the injury site, implanted cells that undergo differentiation generate not only an inert filling tissue, but they are able to stimulate cell recruitment from stem cell niches in order to aesthetically restore the site of injury.