20-Hydroxyecdysone Regulates G Protein-coupled Receptors And Insulin Pathway To Promote Insect Metamorphosis

Background and unsolved questionsInsects can be divided into complete metamorphic insects and incomplete metamorphic insects.The important agricultural pest Helicoverpa armigera we studied belongs to a kind of completely metamorphic insect.The tissue morphology changes extremely significantly.20-hydroxyecdysone(20E)and insulin like peptides(ILPs)play important roles in the growth and development of insects.Insect 20E signaling pathway and insulin/insulin-like growth factor signaling(IIS)pathway(abbreviated as insulin pathway)have antagonistic effects.The steroid hormone 20E initiates the molting and metamorphosis of insects.20E enters the cells and binds to the nuclear receptor EcR to initiate the transcription of downstream genes and initiate the genomic pathway of 20E.Current studies have found that steroid hormones can bind to receptors on the cell membrane to initiate certain nongenomic pathways such as rapid protein modification,rapid changes in calcium ion levels and other rapid physiological effects,which has been confirmed in mammals,Bombyx mori and Drosophila melanogaster.The G protein-coupled receptor(GPCR)on the cell membrane belongs to a family of proteins with a seven-times transmembrane structure,which transduces various signals including neurotransmitters and hormones on the cell membrane.GPCRs are the largest membrane protein family in humans.Studies have found that there are also multiple GPCRs in insects.Our laboratory has identified multiple GPCRs involved in the non-genomic pathway of 20E.Multiple GPCRs are involved in the signal transduction of the same ligand 20E.The mechanism has not yet been fully elucidated.Insulin receptor(INSR)binds insulin to promote larval growth and maintain normal blood glucose levels.Although steroid hormones such as estrogen and 20E are known to antagonize the function of insulin,the molecular mechanism leading to this antagonism is still unclear.In addition,changes in the insulin pathway can lead to diabetes.Insulin can maintain normal blood glucose levels and steroid hormones can antagonize the function of insulin to increase blood glucose and even induce diabetes.However,these mechanisms have not been fully elucidated.ResultsIn the genome of H.armigera,(?)22 genes encoding classic GPCRs that can act as guanine nucleotide exchange factors(GEF)were identified and classified.And the unannotated GPCRs in the genome were also reclassified.We analyzed the GPCRs differentially expressed in the larval midgut transcriptome during metamorphosis stage compared with the feeding stage,and examined the expression profiles of 11 GPCRs with upregulated expression and 2 GPCRs with downregulated expression during the metamorphic stage in the transcriptome.The results showed that there were great differences in their expression in various tissues and developmental stages.The results showed that their expressions were very different in various tissues and in developmental stages.It was further confirmed that the 6 GPCRs involved in the steroid hormone 20E signal pathway were knockdown by RNA interference,and the larvae showed different phenotypes and delayed pupation or formed small pupae.Knockdown of these GPCRs down regulated the expression of HHR3 in 20E signaling pathway.However,only knockdown of GPCR-PrRPR and Smo reduced the expression of the bifunctional phosphatase(Pten)and the transcription factor Forkhead box O(FoxO),which play roles in the process of 20E regulating metamorphosis.Knockdown of adipokinetic hormone receptor Akhr and 5-hydroxytryptamine receptor Htr upregulated transcription factor Krüppel homolog 1(Kr-h1)expression.Knockdown of these four GPCRs all led to delayed pupation.Knockdown of Frizzled 7(Fzd7)downregulated the expression levels of Wnt and cMyc,resulting in weight loss during pupation.Knockdown of the tachykinin receptor TkR86C did not cause a difference in phenotype.The 20E enzyme immunoassay(20E-EIA)analysis confirmed that PRRPR can bind 20E.These results explain the mechanism by which multiple GPCRs transmit 20E signals through different expression profiles and regulation of different gene expression.20E induces the dephosphorylation of INSR to antagonize the function of the insulin signal pathway.The expression and phosphorylation level of INSR were higher during larval feeding stages,but lower during molting metamorphosis stages.Insulin upregulated INSR expression and phosphorylation,while 20E inhibited INSR expression and induced INSR dephosphorylation.20E upregulated the expression of protein tyrosine phosphatase 1B(PTP1B,encoded by Ptpn1),which dephosphorylated INSR.At the same time,20E upregulated the expression of PTEN and maintained the nuclear localization of the transcription factor FoxO.FoxO located in the nucleus promoted the expression of Ptpnl and inhibited the expression of Insr.Using RNA interference technology to knock down Ptpn1 resulted in maintaining the phosphorylation of INSR and leading to small pupae and earlier pupation by increasing the 20E titer.Knock down of Insr inhibited the growth of larvae and reduced the production of 20E,which led to delayed pupation and the small pupae.Knockdown of Insr also led to accumulation of hemolymph glucose.Taken together,these results indicated that 20E counteracts the insulin pathway by dephosphorylating INSR to stop larval growth and accumulate glucose in the hemolymph.Conclusions and scientific significances1.We identified all classic GPCRs in the genome of H.armigera and classified them to provide a reference for the research on the GPCRs of H.armigera.We found that PRRPR is another receptor of 20E.20E regulated the expression of Pten and FoxO by binding to PRRPR or SMO that cannot bind 20E.This further revealed the mechanism that multiple GPCRs transmit 20E signals through different expressions in tissues and the regulation of different gene expressions.It provided a new target for the research of steroid hormone signal pathway and a new theoretical basis for the control of pests.2.INSR functions to promote insect larval growth and 20E production to reach a high titer during the larval feeding stages.The high 20E titer counteracts INSR function by upregulating PTEN expression,which maintains FoxO's nuclear localization to promote PTP1B expression,resulting in INSR dephosphorylation and repressed INSR expression.The critical titer of 20E stops larval growth,determines the body size,triggers metamorphosis,and causes accumulation of hemolymph glucose for imaginal disc growth.This provides new ideas and basis for further research on the interaction between steroid hormones and insulin,and provides a new target for the control of pests.At the same time,it provides theoretical support for the study of blood glucose metabolism,and can also provide a new experimental model for the study of human diabetes.