Insulin And 20-hydroxyecdyone Counteractively Regulate Phosphoinositide-dependent Kinase-1 Expression For Insect Pupation

Research background and scientific questionsThe insulin signaling pathway is a highly conserved pathway in animals.Many studies have shown that the insulin signal pathway participates in animal growth and development,cell growth,and proliferation.3-Phosphoinositide-dependent kinase-1(PDK1)is a central mediator in insulin signaling pathway.PDK1 plays key roles between phosphoinositide 3-kinase(PI3K)and AKT/protein kinase B.When induced by insulin,PI3K phosphorylates phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol(3,4,5)-trisphosphate(PIP3)to attract PDK1 and AKT/PKB to the plasma membrane through their PH domains that interact with PIP3;thus,PDK1 phosphorylates AKT/PKB.The phosphorylated AKT/PKB then phosphorylates the transcription factor forkhead box O(FoxO)to arrest FoxO in the cytoplasm and repress the transcription activity of FoxO in the nucleus.Thus,FoxO loses the function of negative regulation of target genes,and insulin can then promote proliferation and repress apoptosis in cells.A previous study has shown that repression of the insulin signaling pathway influences the morphology of insects.Repression of insulin/TOR signaling in Drosophila by feeding minocycline suppressed the body growth of the larvae and delayed pupation time.Overexpression of PDK1 increased cell and organ size by activating the Akt protein in Drosophila.However,the involvement and mechanism of PDK1 in insect pupation are unknown.Insect pupation is promoted by 20-Hydroxyecdysone(20E).20E is a steroid hormone that promotes insect molting and metamorphosis.20E combines with its nuclear receptor EcRB1 to form the EcRB1/USP1 heterodimeric transcription complex,which regulates gene expression in the 20E pathway,including hormone receptor 3(HR3)and broad(Br)that initiate metamorphosis,including midgut remodeling.20E can repress FoxO phosphorylation,induce FoxO into the nucleus and bind with its DNA element FoxOBE for BR-Z7 transcription,and then regulate carboxypeptidase A(CPA)expression to promote proteolysis for apolysis during insect molting.20E is produced in the prothoracic gland.Insulin promotes cell proliferation and growth in the entire body.Insulin also promotes insect growth to critical body weight,and the prothoracic gland grows larger to produce more 20E for metamorphosis.Because PDK is a key protein kinase in the insulin signaling pathway,we hypothesized PDK is involved in the regulation of insect metamorphosis by insulin.Insect midgut remodeling,including larval midgut programmed cell death(PCD)and imaginal midgut formation,is a key process during metamorphosis in holometabolous insects.Most holometabolous insects need to undergo midgut remodeling during larva-pupa transition for different food consumption.During midgut remodeling,the regenerative cells in the epithelial tissue of the midgut proliferate to form the adult midgut,and autophagy and apoptosis occur in the larval midgut.In the final stage of pupation,dead midgut cells become the yellow body.The yellow body is digested,and the nutrients are absorbed by the adult midgut.Midgut remodeling is controlled by 20E.However,the mechanism underlying the coordinate regulation of midgut remodeling by insulin and 20E is unclear.ResultsIn our study,we identified that PDK1 and FoxO play key roles in interaction between insulin and 20E by using the lepidopteran insect Helicoverpa armigera as a model.The expression level of PDK1 was higher during the larval feeding stages.PDK1 expression was increased by insulin,but repressed by 20E.Knockdown of PDK1 delayed pupation time,and small pupae were formed.Knockdown of PDK1 also decreased AKT/protein kinase B expression and increased FoxO expression.In addition,knockdown of PDK1 blocked midgut remodeling and decreased 20E levels in the larvae.Injection of the larvae with 20E could overcome the effect of PDK1 knockdown,and midgut remodeling recovered.Overexpression of FoxO in HaEpi cells could not induce apoptosis,but promoted autophagy and repressed cell proliferation.ConclusionAll the result indicated that PDK1 was highly expressed during the larval feeding stage.PDK1 plays critical roles to promote cell proliferation and larval growth via promoting FoxO expression under insulin regulation,thereby producing a high 20E titer along with larval growth.20E represses PDK1 expression in a dose-dependent manner.PDK1 represses FoxO expression,thereby inhibiting cell proliferation and promoting apoptosis.A sufficient 20E titer is the key factor for triggering metamorphosis and midgut apoptosis.Innovation and importance of the researchAfter a series of study,we have found the mechanism underlying the coordinate regulation of insect pupation by these two functionally counteractive hormones.We certified PDK1 was the key gene between insulin and 20E coordinate regulation of insect pupation.Identified that 20E titer was the key of triggering metamorphosis and apoptosis.Adding new theory for insect development,produing new target for pests control.