Conversion of 3T3 fibroblasts into adipose cells: triggering of differentiation by prostaglandin F2alpha and 1-methyl-3-isobutyl xanthine.

Abstract

Green and Kehinde [( ) Cell 1, 113-1161 have isolated clones of Swiss 3T3 fibroblasts that are able to convert to adipose cells. In this paper we report on two chemicals (prostaglandin F2a, 0.1 ug/ml, and 1-methyl-3-isobutyl xanthine, 0.5 mM) that are able to rapidly and irreversibly program the fibroblasts to differentiate into adipose cells. Confluent cultures treated with prostaglandin F2a and insulin for 3-5 days, followed by insulin alone for 7-48 hr, yield numerous adipocyte colonies compared to control dishes and dishes treated with insulin alone. Cells treated with prostaglandin F2a or 1-methyl-3-isobutyl xanthine alone, rinsed, and then exposed to insulin gave similar results, indicating that the continuous presence of the triggering agent is not required to elicit the conversion of the fibroblasts to adipocytes. Agents that raise intracellular levels of 3':5'-cyclic AMP (dibutyryl cyclic AMP, 1.0 mM; 8-bromo-cyclic AMP, 0.5 mM; and prostaglandin El, 0.1 tg/ml) do not trigger the conversion process, suggesting that cyclic AMP may not be the mediator of differentiation in these cells. 8-Bromo-cyclic AMP, however, does induce the cyclic AMP phosphodiesterase (3':5'-cyclic-nucleotide phosphodiesterase; 3':5'-cyclic nucleotide 5'-nucleotidohydrolase; EC 3.1.4.17) in these cells; the induction appears to be mediated by increases in intracellular cyclic AMP levels. These results indicate that this cell line might offer a system for studying the regulation of a type of cellular differentiation.Green and his associates (1-3) have isolated a number of clones from the original stock of Swiss 3T3 fibroblasts that convert to adipose cells under defined culture conditions. This conversion is a type of cellular differentiation which occurs after the cells stop growing, at which time there is an increased incorporation of fatty acid precursors into triglycerides. The conversion is blocked by treatment of growing cultures with bromodeoxyuridine. The differentiated phenotype appears, by electron microscopy, to be morphologically similar to normal adipose cells (2). The conversion involves the formation of numerous fat vacuoles, which coalesce into one large fat vacuole with time. The mature adipocyte does not divide. Insulin stimulates the uptake of glucose and the incorporation of radioactively labeled glucose, acetate, and palmitate into triglycerides, while epinephrine and dibutyryl cyclic AMP decrease the triglyceride content (3). These 3T3 fibroblasts appear to offer an excellent model system for the study of a differentiation process using cell culture.In this paper we report the ability of a natural compound (prostaglandin F2a)(PGF2,) and a synthetic compound (1-methyl-3-isobutyl xanthine) (MeiBu-Xan) to trigger the differentiation process by rapidly and irreversibly programming the fibroblasts to differentiate into adipose cells. MATERIALS AND METHODSThe 3T3-L1 fibroblasts were obtained from Dr. H. Green, Massachusetts Institute of Technology, Cambridge, Mass., and were grown in Dulbecco-Vogt's modified ...

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