Potential Roles Of Insulin And Protein Kinase C Signaling Pathways In Pupal Diapause Of The Cotton Bollworm,Helicoverpa Armigera

Most insect species have evolved a period of developmental arrest?diapause?that enables them to circumvent seasonal periods of adversity.Diapause is characterized by metabolic depression,extremely slown development,and increased resistance to stress.It is usually known that diapause is regulated by various hormones.Pupal diapause is due to reduction of prothoracicotropic hormone?PTTH?secreted from brain and results in a decrease of ecdysone secreted from prothoracic glands in cotton bollworm,Helicoverpa armigera.Diapause is a complex physiological process that requires multiple signaling pathways to participate in,such as insulin signaling and TGF-?signaling pathways.Although it has been reported that TGF-?can also regulate aPKC,it is unknown about molecular mechanisms of pupal diapause regulated by insulin and TGF?R-aPKC signals.Insulin signaling pathway has two independent branches of p-Akt and p-ERK.Insulin activates insulin receptor substrate?IRS?via insulin receptor?InR?,then IRS activates two important pathways:1)activation of protein kinase B?PKB/Akt?via phosphatidylinositol 3 kinase?PI3K?,and regulates downstream target proteins such as FOXO;2)Mitogen-activated protein kinase?MAPK?signaling pathway that regulates cell growth and differentiation through Ras,of which the extracellular regulated protein kinase?ERK?in the MAPK subfamily is the most important.It has been reported that protein levels of p-Akt and p-ERK are significantly higher in brains of diapause-destined pupae than in nondiapause pupae in H.armigera,and that ROS regulate p-Akt via InR cascade.To explain how InR regulates p-Akt and p-ERK via ROS,we find that IRS is a key protein to respond to ROS signaling,and that expression of p-IRS is higher in brains of diapause-destined pupae,similar to that of p-Akt or p-ERK.Overexpression of Har-IRS in HzAm1 cells,p-Akt and p-ERK levels increase,whereas p-Akt and p-ERK levels decrease by dsRNA against IRS,indicating that p-Akt and p-ERK are down-stream targets of IRS.When treatment with InR inhibitor or injection inhibitor into pupae,p-InR,p-IRS,p-Akt,and p-ERK levels decrease significantly,suggesting that p-IRS is regulated by p-InR.We then investigate whether ROS regulate the insulin pathway via the protein phosphorylation,and find that p-InR,p-IRS,p-Akt,and p-ERK levels increase by a treatment with H₂O₂ in HzAm1 cells and injection of PQ into pupae to elevate ROS levels.P-InR,p-IRS,p-Akt,and p-ERK levels can be down-regulated by injection of ROS scavenger NAC,indicating that p-InR,p-IRS,p-Akt,and p-ERK are highly expressed in the brain of diapause pupae by up-regulation of ROS.What is the function of p-Akt and p-ERK in brain of diapause-destined papae?It has been reported on ERK associated with ubiquitin,total ubiquitylation levels are up-regulated by overexpression of IRS and ERK in HzAm1 cells,also by treatment with H₂O₂ or injection of PQ into pupae,suggesting that ERK can increase total levels of ubiquitylation.P-Akt may contribute to promote GLUT4 expression for transporting glucose as previous report.Low metabolic activity is an important character in insect diapause,and PEPCK is a key metabolic regulator to produce pyruvate in glucose metabolism.It has been reported that TGF?RI can regulate the aPKC,and aPKC activates metabolic enzyme PEPCK.Thus,we detect expression of TGF?RI,aPKC,p-aPKC and PEPCK during pupal development,and find that expression levels of TGF?RI,aPKC,p-aPKC,and PEPCK are significantly higher in brains of nondiapause pupae than in diapause-destined pupae.Overexpression of TGF?RI up-regulates aPKC and PEPCK,both of dsRNA against aPKC and aPKC inhibitor G6983 treatment in HzAm1 cells down-regulate PEPCK levels.When inhibitor SB431542 for TGF?RI and G6983 for aPKC are injected into pupae,pupal development is delayed,suggesting that TGF?RI-aPKC-PEPCK pathway is involved in the regulation of development or diapause in insects.FOXO is a transcription factor in insulin signaling pathway,and we find a FOXO downstream protein P21,which regulates cell cycle through CDK2 and CyclinE.Developmental expression of FOXO and P21 mRNAs in brain of diapause-destined pupae are higher than in that of nondiapause-destined pupae.FOXO overexpression can increase P21 mRNA in HzAm1 cells and FOXO inhibitor can reduce P21 mRNA levels.CDK2 and CyclinE mRNA levels are higher in brains of nondiapause pupae,and MTT assay shows that FOXO and P21 is closely corralted with cell viability.Thus,FOXO may regulate activity of cyclin CDK2-CyclinE through P21 for controlling cell cycle.In summary,this paper studied the mechanism of Insulin and TGF?RI-?PKC signaling pathway in the diapause of Helicoverpa armigera.It was found that ROS activated p-IRS through p-InR,and p-IRS further activated p-Akt and p-ERK levels,enhancing their phosphorylation levels in diapause pupae.p-ERK affects the level of ubiquitination,and p-ERK is highly expressed in diapause pupae,It may activate ubiquitination to achieve low levels of metabolic state and then slow development.The study also further found that TGF?RI can regulate PEPCK expression through activation of?PKC to regulate the basal metabolism of Helicoverpa armigera,thus affecting pupae diapause.Finally,it was found that FOXO may regulate the activity of CDK2-CyclinE through P21,thereby affecting the cell cycle,and may affect the development of brains during diapause.