The increase of adipose tissue mass that accompanies obesity is due both to an increase in adipocyte number (hyperplasia) and size (hypertrophy) (Shepherd et al., 1993) (Fig. 1). Hyperplasia results from the recruitment of preadipose cells from a population of pluripotent stem cells that reside in the vascular stroma of adipose tissue. These stem cells have the capacity to undergo commitment to adipose, muscle, bone, or cartilage lineages (Young et al., 1995). Both processes, i.e., commitment and hyperplasia to the adipose lineage, can be mimicked and studied separately by using established stem and preadipocyte cell lines in culture. When treated with BMP4, these multipotent stem cells give rise to preadipocytes that undergo mitotic clonal expansion when treated with appropriate differentiation inducers. Following clonal expansion, the cells then undergo terminal differentiation into adipocytes (Tang et al., 2003a, 2003b). Thus, the developmental pathway can be resolved and studied in distinct steps: 1) adipocyte lineage commitment; 2) mitotic clonal expansion; and 3) terminal differentiation into adipocytes. These steps are discussed below.
Many of the key players in the latter 2 phases of the adipocyte development program, i.e., mitotic clonal expansion and terminal differentiation, have been characterized using the 3T3-L1 and 3T3-F442A preadipocyte cell lines established by Howard Green (Green and Meuth, 1974; Patel and Lane, 1999). Little is known, however, about the factors/genes that trigger the commitment process. The goal of recent research in our laboratory has been to identify the genes and proteins that trigger commitment to the adipocyte lineage. To accomplish this objective, we have employed an immortalized multipotent stem cell line, the C3H-10T1/2 line, which under appropriate conditions in cell culture can commit to the adipose, muscle, bone, or cartilage lineages. Shown in the schema (Fig. 2) is our view of where these different immortalized cell lines fit into the pathway of adipocyte development.
COMMITMENT TO THE ADIPOCYTE LINEAGE
We recently identified a factor and developed conditions that cause 10T1/2 stem cells to commit to the adipocyte lineage in cell culture. Moreover, this process can be recapitulated in vivo. Treatment of 10T1/2 stem cells with our usual adipocyte differentiation protocol does not provoke commitment or differentiation, as indicated by failure to express cytoplasmic triglyceride or adipocyte markers. Only after initial exposure to BMP4, followed by treatment with differentiation inducers, do C3H10T1/2 stem cells enter the adipose development pathway and give rise to cells that express the adipocyte phenotype (Tang et al., 2004). We interpret this dependence on BMP4 as evidence for commitment. 3T3-L1 and 3T3-F442A preadipocytes, which are already committed to the adipose lineage, differentiate when subjected to this differentiation protocol in the absence of BMP4. We have been able to verify these findings in an in vivo context. Thus, when 10T1/2 stem cells are first treated with BMP4 in cell culture and then implanted subcutaneously (at a site lacking adipose tissue) into athymic mice, the implanted cells develop into tissue indistinguishable from adipose tissue in normal fat depots of the same animal (Tang et al., 2004).
MITOTIC CLONAL EXPANSION
Differentiation of growth-arrested 3T3-L1 preadipocytes is induced by treatment with a mixture of differentiation inducers, including methylisobutylxanthine, which increases the cellular cAMP level; a glucocorticoid, i.e., dexamethasone; and IGF-1 (or high levels of insulin, which acts through the IGF-1 receptor). This treatment initiates a cascade of events including the synchronous reentry of the cell cycle for 2 rounds of mitosis, a process referred to as mitotic clonal expansion. Preadipocytes traverse the G1/S checkpoint synchronously, as evidenced by: 1) the expression/activation of cdk2-cyclin E/A; 2) downregulation of p27/kip1 (a cdk2-cyclinA inhibitor); and 3) cdk2-cyclinE/A-catalyzed hyperphosphorylation of Rb, which releases E2F from inhibitory constraint and thereby, the transcriptional activation of factors necessary for DNA replication and progression of the cell cycle (Tang et al., 2003b). The events that occur following induction of differentiation until terminal differentiation is completed are illustrated in Figure 3.
Recent evidence shows that mitotic clonal expansion is required for terminal adipocyte differentiation (Tang et al., 2003b; Patel and Lane, 1999, 2000; Zhang et al., 2004). Furthermore, C/EBPβ, a B-Zip transcription factor that is expressed prior to mitotic clonal expansion, plays an essential role in this process (Tang et al., 2003a) and in subsequent events of the differentiation program (Tang et al., 1999).
TERMINAL DIFFERENTIATION PROGRAM
C/EBPβ appears to serve 2 functions, both as an initiator of mitotic clonal expansion and later in the differentiation program, as a transcriptional activator of 2 key adipogenic transcription factor genes, i.e., the C/EBPα and PPARγ genes. C/EBPβ is expressed immediately upon induction of differentiation but is unable to bind DNA (Tang et al., 1999a) and thus cannot yet function as a transcriptional activator. Acquisition of DNA binding activity is delayed until the cells traverse the G1-S checkpoint of the cell cycle, at which point DNA binding activity is acquired. The coincidence of the acquisition of DNA binding activity by C/EBPβ at the G1-S checkpoint is consistent with the dependence of mitotic clonal expansion on this factor. The mechanism of action of C/EBPβ at this point in the program is not known.
Following mitotic clonal expansion, C/EBPβ initiates a cascade of transcriptional activation (Yeh et al., 1995). C/EBPβ activates expression of the C/EBPα and PPARγ genes, which function together as pleiotropic transcriptional activators of the large group of genes that produce the adipocyte phenotype (MacDougald and Lane, 1995; Hwang et al., 1997). Both of the C/EBPα and PPARγ genes possess cis-C/EBP regulatory elements in their proximal promoters, at which C/EBPβ binds and coordinately activates transcription (Christy et al., 1991; Zhu et al., 1995; Tang et al., 1999). Once expressed, C/EBPα is thought to maintain expression of both the C/EBPα and PPARγ genes via transactivation mediated by their respective C/EBP regulatory elements (Christy et al., 1991; Zhu et al., 1995; Tang et al., 1999). Together, C/EBPα and PPARγ function as pleiotropic transcriptional activators of numerous adipocyte genes whose expression produces the differentiated phenotype.