Circadian Influence To Metabolism And Relative Zeitgeber Mechanism Research In Bombyx Mori

Circadian clock is an inner spontaneous timing mechanism of organism which is adapted to the cyclical long-term illumination,temperature environment factor caused by rotation and revolution of earth.Circadian clock is composed by the input pathway,the center pacemaker or oscillator,the output pathway.Input pathway sense the zeitgeber signal and transfer to the central pacemaker,which then arise molecular oscillation through translation and transcription modified.At last the output pathway regulates downstream processes including metabolism through molecular control.Accompanied by circadian fluctuations of energy demand,physiological metabolism also shows the corresponding vibration changes to adapt to the body needs.We used Bombyx mori,which is a model organism of Lepidoptera,to investigate the relation among zeitgeber,circadian pacemaker and metabolism and discuss their machinism.We give zeitgeber signal to the bombyx mori or the BmN cell lines,and established cell lines with the core circadian genes knockdown.Then we use methods such as metabonomics,measurement of enzyme activity and gene expression,and bioinformatics analysis to validate the change of metabolic pathways and analyzed the influence of the biological clock on metabolic mechanism.We also Use immunofluorescence and real-time quantitative polymerase chain reaction(PCR)to verify the predicted molecular clock pathways of Bombyx.The main results were as follows:(1)Zeitgebers influence metabolic pathways through parental factors in fertilization periodParental transcript legacy has an important role in fertilization and development of the early embryo.Parental environmental exposure affects the fertilization of offspring via the transcript legacy,but the underlying biochemical metabolism is largely unresolved.In this study,the parental environmental effects on fertilization of offspring were tested in the silkworm Bombyx mori,an ideal lepidopteran model animal.The results showed the rate of fertilization was decreased after the parents were exposed to a poor environment at 32°C with continuous illumination for 72 h on days 6–9 of the pupal stage,which is a key period for germ cell maturation.This was likely attributable to lower energy charge values,obstructed nicotinamide adenine dinucleotide(NAD~+)regeneration and inactive tricarboxylic acid cycle(TCA)leading to accumulation of large amounts of pyruvic acid and lactic acid.This effect was related to energy metabolism via glycolysis;in particular disruption of the pyruvate metabolic steps.In conclusion,this study showed parental exposure to an abnormal environment during germ cell maturation affected glycolysis and the subsequent fertilization of offspring via the transcript legacy in B.mori.(2)Inhibition of Period or cryptochrome 1 expression causes metabolic abnormalities in BmN cellsAbnormalities in the circadian clock system are known to affect the body's metabolic functions,though the molecular mechanisms responsible remain uncertain.In this study,we achieved continuous knockdown of B.mori Period(BmPer)gene expression in the B.mori ovary cell line(BmN),and generated a Per-KD B.mori model with developmental disorders including small individual cells and slow growth.We conducted cell metabolomics assays by gas chromatography/liquid chromatography-mass spectrometry and showed that knockdown of BmPer gene expression resulted in significant inhibition of glycometabolism.Amino acids that used glucose metabolites as a source were also down-regulated,while lipid metabolism and nucleotide metabolism were significantly up-regulated.Metabolite correlation analysis showed that pyruvate and lactate were closely related to glycometabolism,as well as to metabolites such as aspartate,alanine,and xanthine in other pathways.Further validation experiments showed that the activities of the key enzymes of glucose metabolism,hexokinase,phosphofructokinase,and citrate synthase,were significantly decreased and transcription of their encoding genes,as well as that of pyruvate kinase,were also significantly down-regulated.We concluded that inhibition of the circadian clock gene BmPer repressed glycometabolism,and may be associated with changes in cellular amino acid metabolism,and in cell growth and development.We constructed a victor to express BmCry1_dsRNA,lead to continues knockdown cryptochrome gene(BmCry1)in BmN cells,further obtained developmental disorders Cry1-KD cell line with rapid growth and smaller size.According to the results of metabonomics,glucose-6-phosphoric acid and pyruvate levels showed a significant improve in glycometabolism in the Cry1-KD cell line without the effect of endocrine hormone.And amino acid level which is produced by intermediate of glycometabolism also has a rise trend.Meanwhile,nucleotide metabolism pathways also increased significantly.The correlation analysis of metabolites showed that glucose-6-phosphoric acid and pyruvic acid was closely related to the glycometabolism,amino acid metabolism and nucleotide metabolism pathways.Further validation results show that the activity and expression level of Hk,Pk and Pfk,the key enzymes in glycolysis pathway was changed,and the variation tendency was consistant with the changes in glucose metabolism after BmCry1 gene knock down.The conclusion is that inhibit the expression of circadian clock genes BmCry1 presented the overall abnormal metabolic problems,and improved the glycometabolism of cells,this may be related to the expression levels of key enzymes in metabolic pathways and related enzyme activity.Futher more,the Per-KD and Cry1-KD cell lines were compared and similarities and differences were found.The similarities were smaller size and rised nucleotide metabolism.But their change in glycometabolism,amino acid metabolism and the cell growth speed were almost in contrast.(3)Illumination and termperature exist different oscillator pathways in Bombyx mori cell linesWe use Cry1-KD,Cry2-KD and Per-KD BmN cell lines as material to verify the predicted circadian molecular pathways of Bombyx mori with qPCR and immunohistochemical experiment.The result shows that BmCRY1 could sense the light signal and deliver it to BmPER,which then transfer into the nucleus with the assistance of BmCRY2 protein.Meanwhile,BmTim also transfer into the nucleus with the assistance of BmCRY2.The result of temperature zeitgeber also use Cry1-KD,Cry2-KD and Per-KD BmN cell lines,and the result shows that BmCRY2 might play an important role in both signal sense and feedback regulation in the circadian center pacemaker under temperature zeigeber which is deffer from that under illumination zeigeber.