Expression of growth hormone-independent adipogenesis by a 3T3 cell variant

We have examined the regulation of adipogenesis of a 3T3-F442A cell variant. The variant, designated 3T3-GH-independent clone 16 (GI-16), was isolated after serum-induced adipogenic commitment. 3T3-GI-16 fibroblasts displayed a lower serum requirement for adipogenesis than the 3T3-F442A parent cell. Insulin-stimulated adipogenesis of 3T3-GI-16 cells in serum-free medium (SFM) was extensive in the absence of GH, as judged by oil red O staining or glycerol-3-phosphate-dehydrogenase activity, a property not associated with the 3T3-F442A cell. In SFM devoid of GH the concentration of insulin required to promote half-maximal adipogenesis of 3T3-GI-16 fibroblasts was 5 nM. The expression of GH-independent adipogenesis by 3T3-GI-16 cells was not due to exposure to adipogenic stimuli during routine passage, as insulin-stimulated differentiation was not a function of the inoculation density in nonadipogenic cat serum. We noted that nine proteins resolved by polyacrylamide gel electrophoresis behaved in a differentiation-dependent manner during adipogenesis of 3T3-GI-16 and 3T3-F442A fibroblasts in SFM. The concentrations of all nine proteins were regulated in a GH-independent manner during insulin-stimulated adipogenesis of 3T3-GI-16 fibroblasts. In contrast, the presence of insulin alone markedly altered the expression of only two of the proteins during differentiation of 3T3-F442A cells. The observed changes in the expression of five presently uncharacterized differentiation-dependent proteins were most likely due to employment of SFM. Our results suggest that expression of GH-independent insulin-induced adipogenesis of 3T3-GI-16 fibroblasts reflects a prior commitment by GH during our selection protocol. These results are discussed in the context of a model in which adipogenesis in vivo is postulated to proceed through the sequential action of GH and insulin on target cells.