DNA Synthesis and Mitotic Clonal Expansion Is Not a Required Step for 3T3-L1 Preadipocyte Differentiation into Adipocytes

Abstract

Upon differentiation induction of 3T3-L1 preadipocytes by a hormone mixture containing 1-isobutyl-3-methylxanthine, dexamethasone, and insulin, the preadipocytes undergo ϳ2 rounds of mitotic clonal expansion, which just precedes the adipogenic gene expression program and has been thought to be an essential early step for differentiation initiation. By inducing 3T3-L1 preadipocytes with each individual hormone, it was determined that the mitotic clonal expansion was induced only by insulin and not by 1-isobutyl-3-methylxanthine or dexamethasone. Cell number counting and fluorescence-activated cell-sorting analysis indicated that a significant fraction of 3T3-L1 preadipocytes differentiated into adipocytes without mitotic clonal expansion when induced with the combination of 1-isobutyl-3-methylxanthine and dexamethasone. Furthermore, when normally induced 3T3-L1 preadipocytes were treated with PD98059 (an inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1) to block the activation of extracellular signalregulated kinase (Erk) 1 and Erk2, the mitotic clonal expansion was blocked, but adipocyte differentiation was not affected. These observations were confirmed by bromodeoxyuridine labeling. The differentiated adipocytes induced with 1-isobutyl-3-methylxanthine and dexamethasone or standard hormone mixture plus PD98059 were not labeled by bromodeoxyuridine. Thus, it is evident that 3T3-L1 preadipocytes could differentiate into adipocytes without DNA synthesis and mitotic clonal expansion. Our results also suggested that activation of Erk1 and Erk2 is essential to but not sufficient for induction of mitotic clonal expansion.Obesity has become a major health hazard in many countries and has been indicated as a risk factor for many physiological disorders, such as diabetes, hypertension, and heart problems.As the major cellular component in adipose tissue, adipocytes play a key role in obesity. The excessive growth of adipose tissue in obesity has been suggested as expansion of adipocytes both in cell size and in cell number (1, 2). To better understand adipocyte physiology, in vitro cell models, such as 3T3-L1 and 3T3-F442A, have been used. These model cell lines provide a useful tool to study the adipocyte differentiation process (1, 3). 3T3-L1 cells can be induced to differentiate into mature adipocytes in cell culture (4 -6). It is one of the most used preadipocyte models to study the adipogenesis process. The preadipocyte differentiation program involves several stages. Immediately after hormonal stimulation (IGF-I 1 or insulin at a nonphysiologically high concentration, a glucocorticoid, dexamethasone (DEX), and a cAMP phosphodiesterase inhibitor that increases intracellular cAMP, 3-isobutyl-1-methylxanthine (MIX)), postconfluent G 0 3T3-L1 preadipocytes reenter a period of the cell cycle called mitotic clonal expansion. The gene expression program leading to terminal adipocyte differentiation is initiated during and after this mitotic clonal expansion period. It has been ...

Adipocyte functions are modulated by cell size change: potential involvement of an integrin/ERK signalling pathway

“…38,39 The present study suggests that in mature adipocytes this signalling pathway, together with others, plays a role in the adaptation of fat cell metabolism to change in cell size. It is well established that activation of ERKs results in both transcriptional and posttransductional effects.…”

Grape-seed derived procyanidins interfere with adipogenesis of 3T3-L1 cells at the onset of differentiation

“…36,[39][40][41] Most of the cell cycle related genes in our microarray were downregulated, which suggests that GSPE-treated cells were not Antiadipogenic effects of procyanidins M Pinent et al in the mitotic clonal expansion phase. Opinions on whether mitotic clonal expansion is necessary to allow adipose differentiation are controversial, 42 but recent studies support the idea that mitotic clonal expansion is required for adipogenesis. 43 In fact, Tang et al 44 demonstrated that treatment with PD98059, an inhibitor of MEK (MAPK/ extracellular signal-regulated kinase kinase), delayed mitotic clonal expansion and that this delay was proportional to decreases in differentiation markers and triacylglyceride accumulation.…”

Mitotic clonal expansion: A synchronous process required for adipogenesis

“…In a recent report, Qiu et al (6) concluded that DNA synthesis and MCE are not required for differentiation of 3T3-L1 preadipocytes into adipocytes. In view of considerable circumstantial evidence to the contrary (29,33), were subjected to SDS͞PAGE and immunoblotting as described in Fig.…”

“…Treatment of growth-arrested 3T3-L1 preadipocytes with differentiation inducers initiates mitotic clonal expansion (MCE) before expression of genes that give rise to the adipocyte phenotype (3)(4)(5). Whether MCE is a prerequisite for terminal differentiation remains controversial (6), because definitive proof is lacking. This issue is addressed in the present investigation.…”