A clonal analysis of the differentiation of 3T3‐L1 preadipose cells: Role of insulin

Abstract

Cells of the established preadipose line, 3T3-L1, appear to be undifferentiated fibroblasts during exponential growth. When cells become quiescent, a small percentage of them accumulate triglyceride and become morphologically indistinguishable from mature adipocytes. When insulin is added to quiescent cultures, up to 50% of the cells differentiate into adipocytes. The distribution of lipid-containing cells which appear in clusters of varying sizes was analyzed to determine whether commitment to differentiation occurred after quiescence or during exponential growth and whether insulin was required as an inducer of commitment. The spatial arrangement of 3T3-L1 cells at quiescence on some culture dishes was destroyed by replating. This resulted in random distribution of these cells. The distribution of adipocytes among replated and nonreplated cells in these experiments was compared to a computer generated random distribution of differentiated among undifferentiated cells. Dispersal of cells at confluence resulted in a distribution of fat among nonfat cells not significantly different from the computer generated random distribution. In undisturbed cultures, the distribution of fat cells is not random and is consistent with a commitment event in single cells at any cell division during exponential growth followed by divisions of both committed and uncommitted cells. Since insulin affected the number of mature adipocytes only when added after cessation of exponential growth, insulin is not the inducer of commitment but merely enhances lipid production in previously committed cells.

Staurosporine rapidly commits 3T3‐F442A cells to the formation of adipocytes by activation of GSK‐3β and mobilization of calcium

“…After commitment, clonal expansion involves the activation/translocation of C/EBPb transcription factor, which probably is also present in cells during commitment or part of it. Clonal expansion is positively modulated by IGF-1 Zezulak and Green, 1985], insulin [Steinberg and Brownstein, 1982;Castro-Muñozledo et al, 2003] and L-T 3 [Flores-Delgado et al, 1987]. After clonal expansion, expression of the adipose phenotype begins, coordinated by transcription factors such as PPARg and C/EBPa [Tontonoz et al, 1994;Hu et al, 1995;Zhu et al, 1995;Spiegelman, 1998;Tang et al, 1999]; and modulated by insulin and L-T 3 , among others.…”

“…Prostaglandin F2a [Russell and Ho, 1976], prostacyclin [Négrel et al, 1989], 1-methyl-3-isobutyl xanthine (MIX) [Russell and Ho, 1976], dexamethasone (DEX) [Rubin et al, 1978] or indomethacin [Williams and Polakis, 1977] accelerate the expression of the adipose phenotype. Insulin, IGF-1 and L-T3 increase clonal expansion and regulate the lipogenic enzyme activities of terminally differentiated adipocytes [Steinberg and Brownstein, 1982;Zezulak and Green, 1985;Flores-Delgado et al, 1987;Guller et al, 1988;Dani et al, 1989], whereas growth hormone (GH) [Morikawa et al, 1982[Morikawa et al, , 1984Nixon and Green, 1984] and serum adipogenic factors [Kuri-Harcuch and Green, 1978;Hauner and Lö ffler, 1986;Kuri-Harcuch, unpublished results], seem to be involved in promoting commitment [Castro-Muñ ozledo et al, 2003]. Interferon-g [Keay and Grossberg, 1980], retinoids [Murray and Russell, 1980;Kuri-Harcuch, 1982;Castro-Muñ ozledo et al, 1987], TGF-b [Ignotz and Massague, 1985], and TNFa [CastroMuñ ozledo et al, 2003] inhibit adipocyte differentiation.…”

Establishment and characteristics of porcine preadipocyte cell lines derived from mature adipocytes

“…3E). Steinberg and Brownstein [1982] have suggested that INS is not an inducer of adipocyte differentiation but merely enhances lipid synthesis in cells committed to differentiation. DEX, a synthetic glucocorticoid agonist, strongly stimulates differentiation at early stages in cultured preadipocytes.…”

Differentiation of cultured pre-adipose cells: A probability model

“…if the number of cells in any clone is determined by the number of cell divisions from commitment to confluence. In both the programmed and stochastic models it is assumed that commitment occurs only during exponential growth, a n assumption which is strongly supported by our previous work (Steinberg and Brownstein, 1982). In all models a final clonal pattern exists at confluence before there is any apparent accumulation of lipid.…”

“…In a previous paper we tested a clonal hypothesis of commitment and differentiation and concluded that (1) commitment to fat production occurs prior to the appearance of differentiated cells and that insulin is not an inducer of differentiation but merely a trigger or an enhancer of fat production in previously committed cells; and (2) the pattern of fat among nonfat cells is not compatible with a random distribution but rather is consistent with a clonal origin of fat cell clusters (Steinberg and Brownstein, 1982).…”