PPARγ1 synthesis and adipogenesis in C3H10T1/2 cells depends on S-phase progression, but does not require mitotic clonal expansion

Adipogenesis is typically stimulated in mouse embryo fibroblast (MEF) lines by a standard hormonal combination of insulin (I),dexamethasone(D), and methylisobutylxanthine(M), administered with afresh serum renewal. In C3H10T1/2 (10T1/2) cells, peroxisome proliferator-activated receptor gamma1 (PPARgamma1) expression, an early phase key adipogenic regulator, is optimal after 36 h of IDM stimulation. Although previous studies provide evidence that Mitotic clonal expansion of 3T3-L1 cells is essential for adipogenesis, we show, here, that 10T1/2 cells do not require mitotic clonal expansion, but depend on cell cycle progression through S-phase to commit to adipocyte differentiation. Exclusion of two major mitogenic stimuli (DM without insulin and fresh serum renewal) from standard IDM protocol removed mitotic clonal expansion, but sustained equivalent PPARgamma1 synthesis and lipogenesis. Different S-phase inhibitors (aphidicolin, hydroxyurea, L-Mimosine, and roscovitin) each arrested cells in S-phase, under hormonal stimulation, and completely blocked PPARgamma1 synthesis and lipogenesis. However, G2/M inhibitors effected G2/M accumulation of IDM stimulated cells and prevented mitosis, but fully sustained PPARgamma1 synthesis and lipogenesis. DM stimulation with or without fresh serum renewal elevated DNA synthesis in a proportion of cells (measured by BrdU labeling) and accumulation of cell cycle progression in G2/M-phase without complete mitosis. By contrast, standard IDM treatments with fresh serum renewal caused elevated DNA synthesis and mitotic clonal expansion while achieved equivalent level of adipogenesis. At most, one-half of the 10T1/2 mixed cell population differentiated to mature adipocytes, even when clonally isolated. PPARgamma was exclusively expressed in the cells that contained lipid droplets. IDM stimulated comparable PPARgamma1 synthesis and lipogenesis in isolated cells at low cell density (LD) culture, but in about half of the cells and with sensitivity to G1/S, but not G2/M inhibitors. importantly, growth arrest occurred in all differentiating cells, while continuous mitotic clonal expansion occurred in non-differentiating cells. Irrespective of confluence level, 10T1/2 cells differentiate after progression through S-phase, where adipogenic commitment induced by IDM stimulation is a prerequisite for PPARgamma synthesis and subsequent adipocyte differentiation.