20-hydroxyecdysone Reprogrammes Glucose And Amino Acid Metabolism To Regulate Insect Metamorphosis

Background and scientific questionsHolometabolous insect undergo four developmental stages in their life:egg,larva,pupa and adult.Feeding supports growth and development in larval stages,stops feeding in metamorphosis development stages(from the last larva through pupa to adult),and survive and develop adult tissues by breaking down larval tissue,a process known as autophagy.20hydroxyecdysone(20E)is a crucial hormone that controls insect metamorphosis.The high-titer 20E during metamorphosis inhibits feeding behavior and opposes the insulin signaling pathway,resulting in great changes in the metabolism,from food intake to energy acquisition by decomposing larval tissues,which involves changes in genes transcription and metabolites.In this paper,the major agricultural pest Helicoverpa armigera,cotton bollworm,was used as the research model to investigate the molecular mechanism of 20E regulating metabolic reprogramming during metamorphosis,including the regulation of 20E on KLF expression and its role in autophagy and gluconogenesis;the molecular mechanism of 20E regulating amino acid metabolic reprogramming by metabonomics.ResultsBy detecting the expression changes of KLF 15 during the development and metamorphosis,we found that KLF 15 was highly expressed in the fat body during metamorphosis,and 20E promoted Klf15 transcription through EcR.By observing the morphology of fat body and knockdown experiments,KLF 15 was crucial for the decomposition of fat body during metamorphosis.After knockdown Kf15,a series of metabolites and related genes were detected,and KLF15 was found to promote gluconogenesis,autophagy and apoptosis.The decomposition of fat body was inhibited after knockdown autophagy related gene 8(A tg8),the level of glycogenic substrates including free fatty acids(FFAs),glycerol,and free amino acids(FAAs)decreased,and the glucose levels decreased,indicating that KLF 15 promoted autophagy and provided substrates for gluconogenesis.We found the binding sites of.KLF 15(CACCC,KLF bs)in the promoter sequence of Atg8 and Pepck,and 20E induced the binding of KLF15 to KLF bs to promote the transcription of Atg8 and Pepck.20E reprogrammed glucose metabolism from glycolysis in feeding phase to gluconogenesis in metamorphosis phase.The expression of genes related to glucose metabolism changed significantly,going from high expression of glycolysis,glycogen and trehalose synthase genes during feeding stages to high expression of gluconogenesis,glycogen and trehalose decomposition genes during metamorphosis,leading to the increase of hemolymph glucose levels.These finding showed that 20E promoted KLF15 to integrate autophagy and gluconogenesis to maintain glucose homeostasis during metamorphosis.The metabolites were measured in the hemolymph at the larval growing stage(6th-24 h),wandering stage(6th-96 h)and pupal stage(P6).It was found that the metabolites changed significantly during the development and metamorphosis of H.armigera.Arginine,αketoglutarate(α-KG)and glutamate(Glu)were screened as the mark metabolites of the feeding larva,wandering larva and pupa.Arginine promoted larval cell proliferation,α-KG promoted larval cell autophagy,and Glu promoted adult cell proliferation.By inhibiting the expression of argininosuccinate(Ass),20E inhibited the synthesis of arginine;additionally,by promoting the expression of arginase(Arg),20E promoted the decomposition of arginine,resulting in a significant decrease in arginine levels in the hemolymph after entering the metamorphosis stages compared to the feeding stages.Glutamate dehydrogenase(GDH)was highly expressed in the midgut during feeding and wandering stages,which promoted the conversion of Glu obtained from food into α-KG and increased the le vels of α-KG during wandering stages.After entering the pupal stage,α-KG is transformed into Glu by another glutamate dehydrogenaselike(GDH-like),and FAAs produced by autophagy during metamorphosis were also transformed into Glu,resulting in a significant increase of Glu in the pupal hemolymph,it becomes the primary source of energy for adult development.20E reprogrammed amino acid metabolism to promote tissue remodeling during metamorphosis by regulating the expression of key genes in different metabolic pathways.ConclusionKLF 15 integrated autophagy and gluconeogenesis to maintain glucose homeostasis under 20-hydroxyecdysone regulation1.20E promoted Klf15 expression through EcR.2.KLF 15 promoted Atg8 transcription by binding to KLF bs in the Atg8 promoter,thereby promoting autophagy and apoptosis in the fat body during metamorphosis.3.Autophagy and apoptosis of larva tissue during metamorphosis provided the substrates for gluconeogenesis.4.20E reprogrammed glucose metabolism by regulate the expression of glucose metabolism genes.20-hydroxyecdysone reprogramming amino acid metabolism support the metamorphic development of insects1.The hemolymph metabolites were altered during metamorphosis of Helicoverpa armigera.2.Arginine,α-KG and Glu are abundant marker metabolites of the feeding larvae,wandering larvae and pupae,which promoted larval cell proliferation and growth,larval cell autophagy,and imaginal cell proliferation and growth,respectively.3.The arginine levels were decreased by 20E regulation via repression ofAss expression and upregulation of Arg expression during metamorphosis.4.The α-KG was increased from the transformation of Glu by GDH expressed in the feeding and wandering larval midgut that was repressed by 20E during metamorphosis.The Glu was increased from the transformation of α-KG by GDH-like expressed in the pupal fat body that was upregulated by 20E.5.The FAAs produced from autophagy also contributed to the increased Glu levels at pupal stages.Scientific significanceThis study elucidated the molecular mechanism of steroid hormone 20E reprogramming glucose metabolism and amino acid metabolism by regulating different genes expression,and illustrated the relationship between metabolism and tissue remodeling.It provided new theoretical knowledge for further study of steroid hormone regulation metabolic reprogramming,and provided theoretical basis and target genes for pest control.