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The insulin signal pathway plays an important role in regulating cell growth and cell proliferation.The purpose of this study was to investigate whether the insulin signal pathway is involved in endomitotic DNA replication of silk gland cells.Our results showed that the insulin activated the DNA synthesis.And the specific inhibitor of the insulin signaling pathway in vertebrates,LY294002,a phosphoinositide 3-kinase (PI3K) inhibitor,Rapamycin,a specific inhibitor of mammalian target of rapamycin (TOR),and U0126,the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor,could inhibit the DNA synthesis of silk gland cells by time- and dose-dependent manner,indicating involvement of the PI3K,TOR and MAPK signaling pathways.In vitro treatment,the DNA synthesis induced by insulin was blocked by either LY294002 or Rapamycin.Akt phosphorylation in the silk glands could be induced by the insulin,which was inhibited by LY294002.Phosphorylation of S6K and 4E-BP was stimulated by insulin,and Phosphorylation of S6K induced by insulin was inhibited by Rapamycin,indicating that insulin-stimulated the DNA synthesis is mediated by PI3K,Akt and TOR.Moreover,PI3K and Akt are upstream of TOR in the silk glands.Surprisingly,U0126 did not obviously inhibit the activation by the insulin,and ERK phosphorylation was not stimulated by insulin.So,PI3K/Akt/TOR signaling,which could be induced by insulin,and MAPK/ERK signaling may be two separate signaling pathways in the regulation of DNA synthesis in silk gland cells.