BackgroundThe regulation of glycolysis and autophagy during feeding and metamorphosis in holometabolous insects is a complex process that is not yet fully understood. Insulin regulates glycolysis during the larval feeding stage, allowing the insects to grow and live. However, during metamorphosis, 20-hydroxyecdysone (20E) takes over and regulates programmed cell death (PCD) in larval tissues, leading to degradation and ultimately enabling the insects to transform into adults. The precise mechanism through which these seemingly contradictory processes are coordinated remains unclear and requires further research. To understand the coordination of glycolysis and autophagy during development, we focused our investigation on the role of 20E and insulin in the regulation of phosphoglycerate kinase 1 (PGK1). We examined the glycolytic substrates and products, PGK1 glycolytic activity, and the posttranslational modification of PGK1 during the development of Helicoverpa armigera from feeding to metamorphosis.ResultsOur findings suggest that the coordination of glycolysis and autophagy during holometabolous insect development is regulated by a balance between 20E and insulin signaling pathways. Glycolysis and PGK1 expression levels were decreased during metamorphosis under the regulation of 20E. Insulin promoted glycolysis and cell proliferation via PGK1 phosphorylation, while 20E dephosphorylated PGK1 via phosphatase and tensin homolog (PTEN) to repress glycolysis. The phosphorylation of PGK1 at Y194 by insulin and its subsequent promotion of glycolysis and cell proliferation were important for tissue growth and differentiation during the feeding stage. However, during metamorphosis, the acetylation of PGK1 by 20E was key in initiating PCD. Knockdown of phosphorylated PGK1 by RNA interference (RNAi) at the feeding stage led to glycolysis suppression and small pupae. Insulin via histone deacetylase 3 (HDAC3) deacetylated PGK1, whereas 20E via acetyltransferase arrest-defective protein 1 (ARD1) induced PGK1 acetylation at K386 to stimulate PCD. Knockdown of acetylated-PGK1 by RNAi at the metamorphic stages led to PCD repression and delayed pupation.ConclusionsThe posttranslational modification of PGK1 determines its functions in cell proliferation and PCD. Insulin and 20E counteractively regulate PGK1 phosphorylation and acetylation to give it dual functions in cell proliferation and PCD.

研究背景及科学问题生物体的生长和代谢同时受生长因子和其他激素的调控,它们的活性由相应的受体介导。昆虫生命成长周期主要受三种激素调控,分别为蜕皮激素（20E）、保幼激素（JH）和胰岛素样生长因子（insulinlikepeptide,Ilps）。其中胰岛素信号通路主要控制成长发育,而20E和JH相互作用控制蜕皮变态信号。胰岛素调控幼虫生长达到临界体重,之后释放促前胸腺激素（PTTH）促进蜕皮激素的合成与释放,最后于组织加工成活性代谢物20E。在果蝇中,insulin由胰岛β细胞合成,刺激组织及PG的生长,使PG分泌大量20E,启动变态发育。果蝇中,20E通过促进转录因子dFOXO（ForkheadboxO,FoxO）的核定位和翻译抑制因子4E-BP的转录,对抗胰岛素促进生长的作用。家蚕中,20E对受体复合物EcRB1/USP1及insulin受体在不同的生理条件下有不同的调控作用。棉铃虫的前期研究结果表明,存在一种细胞周期蛋白依赖性激酶调节亚基1（CKS1）受20E和insulin共同调节,保证虫体生长和变态,insulin上调CKS1表达,而高浓度20E则抑制CKS1表达。另外发现一种GTP酶-Rab4b介入20E和insulin通路的相关基因的转录调控。20E可以上调磷脂酰肌醇-3,4,5-三磷酸3-磷酸酶（Phosphatidylinositol-3,4,5-trisphosphate3-phosphatase,PTEN）和叉头框蛋白FoxO的表达,同时PTEN可以通过抑制AKT磷酸化实现抑制FoxO磷酸化,导致FoxO的核定位,实现对胰岛素途径的拮抗。以上研究均表明20E参与胰岛素途径调节,但尚不清楚具体作用机制。本论文以鳞翅目昆虫棉铃虫为模型,选择胰岛素受体IR为靶标,通过激素刺激、双链RNA干扰、构建过表达质粒等实验技术,研究20E对IR的调控,阐明20E调控胰岛素信号途径的分子机制,为害虫的防治提供理论依据和靶标基因。研究结果及结论虫体双链RNA干扰IR,与对照组比,实验组出现高死亡率、化蛹时间延迟及化小蛹等表型。虫体干扰IR后检测到胰岛素通路下游相关基因受到表达抑制。在虫体的发育阶段,IR表达量在取食期高,蜕皮变态期下降。虫体组织和棉铃虫表皮细胞系上,insulin以浓度和时间依赖的方式上调IR表达。而20E则是依赖浓度梯度双向调节IR表达,低浓度20E促进IR表达,高浓度20E抑制IR表达。在HaEpi细胞系上叠加insulin和不同浓度的20E刺激后得到相同结果,低浓度20E促进insulin诱导的IR表达和磷酸化,而高浓度20E抑制insulin诱导的IR表达和磷酸化。Ilps在棉铃虫幼虫到蛹的发育时期均有表达,并且在末龄幼虫和蛹期呈现上调表达趋势,说明末龄幼虫和蛹期20E不是通过抑制IPs的表达来抑制IR磷酸化的。放线菌酮及专门抑制磷酸酶的一种抑制剂预处理细胞后发现高浓度20E抑制IR磷酸化现象减弱,意味着高浓度20E使IR发生去磷酸化是通过上调一种磷酸酶完成。在细胞系干扰PTEN后,高浓度20E抑制IR磷酸化效果减弱,单独过表达PTEN可以引起IR去磷酸化,说明高浓度20E通过上调PTEN使IR去磷酸化,但没有检测到20E诱导下PTEN与IR互作,推测PTEN间接调控IR磷酸化。研究得出如下结论:胰岛素信号通路由IR介导调节幼虫生长和发育,insulin上调控IR表达和磷酸化。高浓度20E促进Ips表达,高浓度20E通过上调PTEN表达抑制IR磷酸化。以上机制研究表明20E基于浓度促进或抑制IR的表达及磷酸化,参与胰岛素信号通路调控。创新性及意义本论文首次发现20E依赖浓度双向调控IR表达和磷酸化参与胰岛素信号通路调控,为20E和胰岛素共同调控棉铃虫生长发育提供了新的数据支持。

The regulation of glycometabolism homeostasis is vital to maintain health and development of animal and humans; however, the molecular mechanisms by which organisms regulate the glucose metabolism homeostasis from a feeding state switching to a non-feeding state are not fully understood. Using the holometabolous lepidopteran insect Helicoverpa armigera, cotton bollworm, as a model, we revealed that the steroid hormone 20-hydroxyecdysone (20E) upregulated the expression of transcription factor Kruppel-like factor (identified as Klf15) to promote macroautophagy/autophagy, apoptosis and gluconeogenesis during metamorphosis. 20E via its nuclear receptor EcR upregulated Klf15 transcription in the fat body during metamorphosis. Knockdown of Klf15 using RNA interference delayed pupation and repressed autophagy and apoptosis of larval fat body during metamorphosis. KLF15 promoted autophagic flux and transiting to apoptosis. KLF15 bound to the KLF binding site (KLF bs) in the promoter of Atg8 (autophagy-related gene 8/LC3) to upregulate Atg8 expression. Knockdown Atg8 reduced free fatty acids (FFAs), glycerol, free amino acids (FAAs) and glucose levels. However, knockdown of Klf15 accumulated FFAs, glycerol, and FAAs. Glycolysis was switched to gluconeogenesis, trehalose and glycogen synthesis were changed to degradation during metamorphosis, which were accompanied by the variation of the related genes expression. KLF15 upregulated phosphoenolpyruvate carboxykinase (Pepck) expression by binding to KLF bs in the Pepck promoter for gluconeogenesis, which utilised FFAs, glycerol, and FAAs directly or indirectly to increase glucose in the hemolymph. Taken together, 20E via KLF15 integrated autophagy and gluconeogenesis by promoting autophagy-related and gluconeogenesis-related genes expression.Author summaryGlucose is the direct substrate for energy production in animal and humans. Autophagy and gluconeogenesis are known to help organisms maintaining energy substrates; however, the mechanism of integration of autophagy and gluconeogenesis is unclear. Holometabolous insects stop feeding during metamorphosis under steroid hormone 20-hydroxyecdysone (20E) regulation, providing a good model for the study. Using lepidopteran insect Helicoverpa armigera, cotton bollworm, as a model, we revealed that Kruppel-like factor 15 (KLF15) integrated autophagy and gluconeogenesis to maintain glucose homeostasis under 20E regulation. 20E increased Klf15 expression, and KLF15 in turn promoted autophagy-related and gluconeogenesis-related genes expression during metamorphosis. Autophagy and apoptosis of the fat body provided substrates for gluconeogenesis. This work clarified the important functions and mechanisms of KLF15 in autophagy and glycometabolism reprogramming for glucose homeostasis after feeding stop during insect metamorphosis.

During development, cells constantly undergo fate choices by differentiating, proliferating, and dying as part of tissue remodeling. However, we only begin to understand the mechanisms of these different fate choices. Here, we took the lepidopteran insect Helicoverpa armigera, the cotton bollworm, as a model to reveal that insulin-like growth factor 2 (IGF-2-like) prevented cell death by promoting cell growth and proliferation. Tissue remodeling occurs during insect metamorphosis from larva to adult under regulation by 20-hydroxyecdysone (20E), a steroid hormone. An unknown insulin-like peptide in the genome of H. armigera was identified as IGF-2-like by sequence analysis using human IGFs. The expression of Igf-2-like was upregulated by 20E. IGF-2-like was localized in the imaginal midgut during tissue remodeling, but not in larval midgut that located nearby. IGF-2-like spread through the fat body during fat body remodeling. Cell proliferation was detected in the imaginal midgut and some fat body cells expressing IGF-2-like. Apoptosis was detected in the larval midgut and some fat body cells that did not express IGF-2-like, suggesting the IGF-2-like was required for cell survival, and IGF-2-like and apoptosis were exclusive, pointing to a survival requirement. Knockdown of Igf-2-like resulted in repression of growth and proliferation of the imaginal midgut and fat body. Our results suggested that IGF-2-like promotes cell growth and proliferation in imaginal tissues, promoting cell death avoidance and survival of imaginal cells during tissue remodeling. It will be interesting to determine whether the mechanism of action of steroid hormones on insulin growth factors is conserved in other species.

The regulation of glycometabolism homeostasis is vital to maintain health and development of animal and humans;however,the molecular mechanisms by which organisms regulate the glucose metabolism homeostasis from a feeding state switching to a non-feeding state are not fully understood.Using the holometabolous lepidopteran insect Helicoverpa armigera,cotton bollworm,as a model,we revealed that the steroid hormone 20-hydroxyecdysone(20E)upregulated the expression of transcription factor Krüppel-like factor(identified as Klf15)to promote macroautophagy/autophagy,apoptosis and gluconeogenesis during metamorphosis.20E via its nuclear receptor EcR upregulated Klf15 transcription in the fat body during metamorphosis.Knockdown of Klf15 using RNA interference delayed pupation and repressed autophagy and apoptosis of larval fat body during metamorphosis.KLF15 promoted autophagic flux and transiting to apoptosis.KLF15 bound to the KLF binding site(KLF bs)in the promoter of Atg8(autophagy-related gene 8/LC3)to upregulate Atg8 expression.Knockdown Atg8 reduced free fatty acids(FFAs),glycerol,free amino acids(FAAs)and glucose levels.However,knockdown of Klf15 accumulated FFAs,glycerol,and FAAs.Glycolysis was switched to gluconeogenesis,trehalose and glycogen synthesis were changed to degradation during metamorphosis,which were accompanied by the variation of the related genes expression.KLF15 upregulated phosphoenolpyruvate carboxykinase(Pepck)expression by binding to KLF bs in the Pepck promoter for gluconeogenesis,which utilised FFAs,glycerol,and FAAs directly or indirectly to increase glucose in the hemolymph.Taken together,20E via KLF15 integrated autophagy and gluconeogenesis by promoting autophagy-related and gluconeogenesis-related genes expression.

Methoprene-tolerant 1 (Met1) is a basic-helix-loop-helix Per/Arnt/Sim (bHLH-PAS) protein identified as the intracellular receptor of juvenile hormone (JH). JH induces phosphorylation of Met1; however, the phosphorylation site and outcomes of phosphorylation are not well characterized. In the present study, using the lepidopteran insect and serious agricultural pest Helicoverpa armigera (cotton bollworm) as a model, we showed that JH III induced threonine-phosphorylation of Met1 at threonine 393 (Thr393) in the Per-Arnt-Sim (PAS) B domain. Thr393-phosphorylation was necessary for Met1 binding to the JH response element (JHRE) to promote the transcription of Kr-h1 (encoding transcription factor Kruppel homolog 1) because Thr393-phosphorylated Met1 increased its interaction with Taiman (Tai) and prevented the Met1-Met1 association. However, JH III could not prevent Met1-Met1 association after Met1-Thr393 was mutated, suggesting that Thr393-phosphorylation is an essential mechanism by which JH prevents Met1-Met1 association. The results showed that JH induces Met1 phosphorylation on Thr393, which prevents Met1-Met1 association, enhances Met1 interaction with Tai, and promotes the binding of Met1-Tai transcription complex to the E-box in the JHRE to regulate Kr-h1 transcription.

The insulin receptor (INSR) binds insulin to promote body growth and maintain normal blood glucose levels. While it is known that steroid hormones such as estrogen and 20-hydroxyecdysone counteract insulin function, the molecular mechanisms responsible for this attenuation remain unclear. In the present study, using the agricultural pest lepidopteran Helicoverpa armigera as a model, we proposed that the steroid hormone 20-hydroxyecdysone (20E) induces dephosphorylation of INSR to counteract insulin function. We observed high expression and phosphorylation of INSR during larval feeding stages that decreased during metamorphosis. Insulin upregulated INSR expression and phosphorylation, whereas 20E repressed INSR expression and induced INSR dephosphorylation in vivo. Protein tyrosine phosphatase 1B (PTP1B, encoded by Ptpn1) dephosphorylated INSR in vivo. PTEN (phosphatase and tensin homolog deleted on chromosome 10) was critical for 20E-induced INSR dephosphorylation by maintaining the transcription factor Forkhead box O (FoxO) in the nucleus, where FoxO promoted Ptpn1 expression and repressed Insr expression. Knockdown of Ptpn1 using RNA interference maintained INSR phosphorylation, increased 20E production, and accelerated pupation. RNA interference of Insr in larvae repressed larval growth, decreased 20E production, delayed pupation, and accumulated hemolymph glucose levels. Taken together, these results suggest that a high 20E titer counteracts the insulin pathway by dephosphorylating INSR to stop larval growth and accumulate glucose in the hemolymph.

Animal steroid hormones initiate signaling by passive diffusion into cells and binding to their nuclear receptors to regulate gene expression. Animal steroid hormones can initiate signaling via G protein-coupled receptors (GPCRs); however, the underlying mechanisms are unclear. Here, we show that a newly discovered ecdysone-responsive GPCR, ErGPCR-3, transmits the steroid hormone 20-hydroxyecdysone (20E) signal by binding 20E and promoting its entry into cells in the lepidopteran insect Helicoverpa armigera. Knockdown of ErGPCR-3 in larvae caused delayed and abnormal pupation, inhibited remodeling of the larval midgut and fat body, and repressed 20E-induced gene expression. Also, 20E induced both the interaction of ErGPCR-3 with G proteins and rapid intracellular increase in calcium, cAMP and protein phosphorylation. ErGPCR-3 was endocytosed by GPCR kinase 2-mediated phosphorylation, and interacted with beta-arrestin-1 and clathrin, to terminate 20E signaling under 20E induction. We found that 20E bound to ErGPCR-3 and induced the ErGPCR-3 homodimer to form a homotetramer, which increased 20E entry into cells. Our study revealed that homotetrameric ErGPCR-3 functions as a cell membrane receptor and increases 20E diffusion into cells to transmit the 20E signal and promote metamorphosis.

Author summaryThe regulatory subunits of phosphatidylinositol 3-kinases (PI3Ks) play very important roles in various pathways by promoting cell proliferation or apoptosis. However, the upstream regulatory mechanism of their opposite functions is unclear. Using a seriously agricultural pest Helicoverpa armigera as a model, we show that ILPs induce HaP60 phosphorylation to increase HaP110 phosphorylation and cell membrane location to promote cell proliferation. 20E promotes HaP60 and HaP110 dephosphorylation that resulted in the cytosol localization and inhibition of PI3K activity. Moreover, 20E elevates HaP60 expression to promote apoptosis. Our study revealed that HaP60 plays dual functions to regulate cell proliferation and apoptosis by changing its phosphorylated status.The regulatory subunits (P60 in insects, P85 in mammals) determine the activation of the catalytic subunits P110 in phosphatidylinositol 3-kinases (PI3Ks) in the insulin pathway for cell proliferation and body growth. However, the regulatory subunits also promote apoptosis via an unclear regulatory mechanism. Using Helicoverpa armigera, an agricultural pest, we showed that H. armigera P60 (HaP60) was phosphorylated under insulin-like peptides (ILPs) regulation at larval growth stages and played roles in the insulin/ insulin-like growth factor (IGF) signaling (IIS) to determine HaP110 phosphorylation and cell membrane translocation; whereas, HaP60 was dephosphorylated and its expression increased under steroid hormone 20-hydroxyecdysone (20E) regulation during metamorphosis. Protein tyrosine phosphatase non-receptor type 6 (HaPTPN6, also named tyrosine-protein phosphatase corkscrew-like isoform X1 in the genome) was upregulated by 20E to dephosphorylate HaP60 and HaP110. 20E blocked HaP60 and HaP110 translocation to the cell membrane and reduced their interaction. The phosphorylated HaP60 mediated a cascade of protein phosphorylation and forkhead box protein O (HaFOXO) cytosol localization in the IIS to promote cell proliferation. However, 20E, via G protein-coupled-receptor-, ecdysone receptor-, and HaFOXO signaling axis, upregulated HaP60 expression, and the non-phosphorylated HaP60 interacted with phosphatase and tensin homolog (HaPTEN) to induce apoptosis. RNA interference-mediated knockdown of HaP60 and HaP110 in larvae repressed larval growth and apoptosis. Thus, HaP60 plays dual functions to promote cell proliferation and apoptosis by changing its phosphorylation status under ILPs and 20E regulation, respectively.