Effects Of 1-DNJ On Key Metabolic Pathways,Such As Glycometabolism, Were Studied By Using Eri Silkworm As A Model

Mulberry leaves have been widely cultivated for rearing the silkworm,B.mori from ancient times,but mulberry trees are not the natural host plants of Eri silkworm,S.cynthia ricini.Mulberry leaves exude latex containing rich defense protein MLX56 and alkaloids,such as DNJ.These active substances are lethal to Eri silkworms and Mamestra brassicae Linnaeus,the generalist herbivorous insect.S.cynthia ricini larvae have been applied to detect and assess the levels of plant defense responses to herbivorous insects.S.cynthia ricini larvae are maintained in our laboratory to evaluate the effects of mulberry latex and DNJ on the herbivorous insects.1-DNJ is a natural D-glucose analogue with promising physiological activity in vivo.It can inhibit ?-glucosidase in the small intestine and the liver,delay the senescence of blood vessels and affect the reverse cholesterol metabolism.Due to the potential medicinal value of DNJ,DNJ-enriched products may represent a therapy or oral treatment of diabetes mellitus,and numerous DNJ tablets have been commercially available in many countries.These studies illustrate the bioavailability of DNJ,but their DNJ content has not been specified.Previous studies focused on the antidiabetic role of DNJ in rats or other mammals,but direct physiological evidences clearly elaborating the detailed physiological mechanisms of glycometabolism or toxicological effects of DNJ against insects were not reported.Nuclear magnetic resonance(NMR)can achieve non-destructive,non-selective and non-biased analysis on samples,which can be carried out under the near-physiological condition,and the experimental methods are also flexible.Metabonomics analysis method that based on NMR is one of the main technologies in current metabolomics,which is suitable for the study of the complex components in metabolites,and also suitable for the pathology research and improving mechanism research after drug action.In this study,metabolomics technology is applied to study the metabolic regulatory mechanism of DNJ in S.cynthia ricini,which is conductive for the illustration of glycometabolism,energy metabolism and other pathways changes caused by DNJ.Meanwhile,transcriptome technology and RT-q PCR validation are also adopted to identify related enzymes and metabolic pathways of S.cynthia ricini glycometabolism and energy metabolism,combining metabolomics data,it analyzes the differential expression of key enzymes genes of catalyzedglycometabolism and energy metabolism after S.cynthia ricini fed with DNJ.The main results are as follows:1.From the four-instar,S.cynthia ricini was fed with 5?l H2 O,0.25% DNJ,0.5%DNJ,latex and a mixture(0.5% DNJ and latex was mixed up with 1: 1),once a day for two days;in the third day,S.cynthia ricini death only occurred in latex group,with the mortality rate of 40%.In order to further verify the toxicity of DNJ to Eri silkworms,the five-instar of S.cynthia ricini was fed with 5?l H2 O,1 and 2% DNJ,once a day for 3 days,none of silkworms died.Experimental results showed that: DNJ has no obvious toxic reaction to S.cynthia ricini in short time,but the unidentified high-molecular-weight factors(UHMWs)and MLX56 are likely the lethal factor of S.cynthia ricini,besides,they also have concentration-dependent.2.Based on the NMR metabonomics technology,we analyzed the alteration of glycometabolism and energy metabolism pathways in hemolymph,midgut and fat body of S.cynthia ricini caused by DNJ.We conducted pattern identification and metabolites quantitative analysis on the obtained data,the result showed that there was significant metabolic profiling difference in hemolymph,midgut and fat body between different concentrations of DNJ group and the control group.Through quantitative analysis,it showed that,trehalose,lactate and alanine concentration increased in hemolymph of DNJ group,while the concentration of the intermediate product in TCA cycle was reduced,indicating that trehalose hydrolysis,gluconeogenesis and TCA cycle level were reduced,glycolytic pathway was enhanced,while the activity of involved catalyzing enzyme was changed.The hydrolysis of trehalose and TCA cycle were inhibited in midgut and fat body by DNJ.3.The DNJ and control group of the five-instar S.cynthia ricini were analyzed by transcriptome technology,the basic information of S.cynthia ricini genome was obtained.The results showed,after adding DNJ,the up-regulared expression of unigenes in S.cynthia ricini were 577,down-regulated expression of unigenes were288.In the meanwhile,we analyzed the differential expression of key enzymes genes of catalyzed glycometabolism and energy metabolism of S.cynthia ricini after oral administration of 2% DNJ.The results showed that,25 unigenes expression of succinate dehydrogenase,enzyme fumarate,malate dehydrogenase and trehalase with possibly changed DNJ group activity remained no difference.The expression of unigenes related to glucosidase were up-regulared,though its enzymatic activity was inhibited.4.In this experiment,relative quantitative PCR method is adopted to measure the m RNA expression of trehalase and glucosidase genes in S.cynthia ricini hemolymph.There are 2 trehalase gengs: the expressions of trehalase-1B and trehalase-2 were no different from the control group after 6-48 h of S.cynthia ricini fed with 1% and 2%DNJ.In 3 glucosidase gengs,?-glucosidase gene was significantly up-regulated after 12 h of S.cynthia ricini fed with 2% DNJ 12 h,while the expression of glucosidase ? ? subunit gene was increased after 6-12 h of S.cynthia ricini fed with 2% DNJ,there was no significant difference between the experimental group and the control group at at different time in regards of uncharacterized family 31 glucosidase gene expression.The experimental result was basically in line with gene differential expression analysis in transcriptome.In summary,the effect of DNJ on glycometabolism and energy metabolism in S.cynthia ricini was obtained in our paper.The results also showed the transcription level difference of the involved key enzyme.It provides a reference for further study of the drug target and the specific mechanism of DNJ.