Insulin signaling in adipocytes differentiated from mouse stromal MC3T3-G2/PA6 cells

The stromal MC3T3-G2/PA6 (PA6) cells from mouse clavaria did not require insulin for differentiation into mature adipose cells, although insulin is well known to play a key role in adipocyte differentiation. Large lipid droplets were observed in the cytoplasm of PA6 cells, and mRNA expression of the adipose specific proteins (aP2, PPARgamma, C/EBPalpha, FAS, GLUT4, leptin, and adiponectin) as differentiation markers appeared or increased clearly in the cells at 8 d after stimulation without insulin. In addition, the glycerol released from the cells (lipolysis) was increased in a concentration-dependent manner by isoproterenol. However, the isoproterenol-induced lipolysis in the cells was not influenced by treatment with insulin, although that was observed in extramedullary adipocytes, 3T3-L1 cells. On the other hand, the 2-deoxy-D-[1-3H]glucose uptake in differentiated PA6 cells also increased by insulin, as shown in other adipose cells. In the cells, insulin induced the phosphorylation of extracellular signal-regulated kinases (Erks), Akt at Ser 473 and ribosomal p70 S6 protein kinase (p70 S6K) at Thr 389, and the insulin-induced 2-deoxy-D-[1-3H]glucose uptake was inhibited by pre-treatment with wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K), or ML-9, an Akt inhibitor. These results suggest that the insulin signal for adipogenesis (lipogenesis) and lipolysis in bone marrow stroma PA6 cells differs from extramedullary adipocytes, such as 3T3-L1 cells.