| Flavin-containing monooxygenases(FMOs),like cytochromes P450s(P450s),were considered as the important phase ? drug metabolizing enzymes,which could participate in the detoxification metabolism of xenobiotics in mammals.However,there were no studies on whether insect FMOs had played a similar role in mediating the insecticides resistance,and their other latent functions were still unknown.Recent studies had shown that the expression levels of FMO3 in mammals were associated with cholesterol balance,diabetes and other metabolic diseases,which implied FMOs might participate in the carbohydrate and lipid metabolism,but its mechanism remained unrecognized.According to the conservative structural domains and only two FMOs genes(Dmfmo1 and Dmfmo2)they had,Drosophila melanogaster,were selected as the model to construct transgenic flies whose mRNA expression were upregulated or downregulated by the GAL4/UAS binary expression system,and to explore the effects of the various expression levels of Dmfmos on the insecticide susceptibilities and carbohydrate and lipid metabolism;meanwhile,the endogenous substrates of FMOs were mined through metabolomics analysis to elucidate the internal mechanism of FMOs in regulating carbohydrate and lipid metabolism.The research contents were as follows:?.Expression levels of Dmfmos could affect the susceptibilities to different insecticides in D.melanogasterAs an important detoxification enzyme,the FMOs mainly participated in the oxidative metabolism of drugs and other xenobiotics in mammals.In contrast,it was not clear whether insect FMOs also had the function of exogenous drugs metabolism,especially for the insecticides,which was of great significance for pest management.Based on the substrate-dependent metabolism characteristics of hFMOs,five insecticides with the different amine structures were selected in this study.The diagnostic bioassay results showed that the most significant difference in susceptibility to cartap between FMOs-OE(gene overexpression)and the control group,the mortality rates were reduced by 66.7%?73.3%;then for the dinotefuran possessing the secondary amine structure,the mortality rates were reduced by 33.3%?26.7%.However,for the clothianidin possessing secondary amine structure,only Dmfmol overexpression could significantly reduce the mortality of the flies(30%);for the nitenpyram possessing secondary and tertiary amine structures,only overexpression of Dmfmo2 could significantly decrease the mortality of flies(36.7%).There were no significant differences in the mortality of Drosophila melanogaster with only tertiary amine structure.Therefore,as a phase ? metabolic enzymes,DmFMOs might decrease toxicities by metabolizing insecticides containing some characteristic amines and had various metabolic capacities for the different amine structures,which also indicated that DmFMOs should be considered as the insect detoxification enzymes which mediated the insecticide resistance.?.Regulation of carbohydrate and lipid metabolism related to the expression levels of flavin-containing monooxygenases in D.melanogasterIn order to prove the hypothesis that insect FMOs was related to the carbohydrate and lipid metabolism in insect,a series of experiments was performed using the fruit flies and Drosophila S2 cells,including the exogenously adding the FMOs competitive inhibitor,cellular RNAi,and inducible GAL4/UAS binary expression system to manipulate gene expressions,to verify the results by detecting the biochemical indexes and the expression levels of gene transcripts related to carbohydrate and lipid metabolism.Due to the high binding affinity,methimazole(MMI)was often used as the competitive inhibitor in FMOs metabolism study.In this study,a series of experiments in vivo(fed with 30 mM methimazole in diet for two weeks)and in vitro(incubated S2 cells with 300 ?M methimazole for 48 h)had been conducted to explore the influences of MMI on carbohydrate and lipid metabolism in fruit flies.The results showed that MMI could significantly reduce triglyceride contents(18.1%in vivo and 30.6%in vitro)and increase glycogen contents(10.7%in vivo and 126.8%in vitro).Meanwhile,Dmfmo1,s6k,dilp2,acc and dilp5 were remarkably downregulated at the levels of mRNA transcription.In addition,the average lipid drop area in S2 cells after adding MMI was significantly reduced by about 25%compared with the control group,which was consistent with the decreased triglyceride content measured at the cellular level.These results demonstrated that methimazole might affect the carbohydrate and lipid metabolism in Drosophila by inhibiting the enzyme activities of DmFMOs.In order to further verify the relationship between DmFMOs and carbohydrate and lipid metabolism,RNA interference was applied in the knockdown of Dmfmos in Drosophila S2 cells.The results indicated that the expression levels of Dmfmo1 and Dmfmo2 were reduced by 88.8%and 82.9%,respectively,compared with the control group(addition of dsegfp)after the incubation with dsRNA for 72 hours.The biochemical assays showed that downregulation of Dmfmos could reduce the triglyceride levels(20.9%?27.3%)and increase the glycogen contents(18.2%?22.2%)in Drosophila S2 cells.Furthermore,oil red O(ORO)staining was performed to evaluate the content of neutral lipids in Drosophila S2 cells,which showed that downregulation of Dmfmol and Dmfmo2 could significantly reduce the lipid droplet content of fixed cells by 21.3%and 44.9%compared to the controls,respectively.At the transcriptional level,knockdown of Dmfmo1 could significantly reduce the expression levels of fas,acc and ilp2(17.5%?47.1%),and upregulate the expression level of hsl(81.0%)compared with the control group.Similarly,downregulated Dmfmo2 mRNA also remarkably decreased the expression levels of fas and acc(32.5%and 40.8%,respectively)and significantly increased the expression levels of ilp5 and hsl(85.7%and 48.7%,respectively).These results were basically consistent with the ones in experiments with the addition of FMOs competitive inhibitors exogenously.Therefore,Dmfmos expression levels could influence carbohydrate and lipid metabolism of Drosophila S2 cells.Based on the results at the cellular level,we also detected the effects of Dmfmos expression levels on carbohydrate and lipid metabolism in Drosophila adults.Compared with the control group,Dmfmo1 genes had increased expression levels of 2847%and 4650%,respectively,in DmFMOs-OE females and males,and decreased expressions of 87.3%and 96.4%,respectively,in DmFMOs-KD(gene knockdown)females and males;Dmfmo2 genes had increased expressions of 343%?482%,respectively,in DmFMOs-OE females and males,and decreased expressions of 63.1%?89.1%,respectively,in DmFMOs-KD females and males.Then for the mid-aged male flies with knockdown or overexpression of Dmfmos,the results showed that downregulated Dmfmos could reduce the content of triglyceride,while upregulated Dmfmo1 could increase the its content.Downregulated Dmfmo1 could significantly reduce the trehalose levels.Upregulated Dmfmos could significantly increase the contents of glycogen and downregulated Dmfmos could reduce the levels of free fatty acid.Meanwhile,the transcript levels of the genes related to carbohydrate and lipid metabolism also had been detected.The results showed that knockdown of Dmfmo1 could reduce the expression levels of fas,acc and ilp2 by 12.0%?36.1%.The transcript levels of ilp5,akh,bmm,lip3 and srebp were significantly increased by 31.1%?113.6%.Similarly,downregulated Dmfmo2 could decrease the expression levels of fas,acc and ilp3 by 40.1%?60.1%,while ilp5,bmm and lip3 were significantly upregulated by 48.6%?198.1%.Taken together,Dmfmos could regulate the carbohydrate and lipid metabolism in Drosophila,which were basically consistent with the results at the cellular level.?.Endogenous metabolic substrate analysis of Drosophila flavin-containing monooxygenasesPrevious studies had demonstrated the relationship between Drosophila FMOs and carbohydrate and lipid metabolism,but how DmFMOs participated in and affected this process or whether there were endogenous substrates in vivo were still unknown.To address these issues,we performed a comparative metabolomic analysis for the Drosophila adults with the downregulation and overexpression of Dmfmos,which could contribute to reveal the potential substrate and functions of DmFMOs.Firstly,multivariate analyses indicated significant differences in metabolic status among the various groups.Then according to the univariate analyses and q-values,comparing with the control group,it showed the significant differences for the levels of phosphatidyl compounds in FMO-KD groups,mainly including the phosphatidyl serine and phosphatidyl inositol(45.08-fold?131.1-fold increased),and phosphatidyl choline,phosphatidyl ethanolamine,and phosphatidyl glycerol(0.1312-fold?0.3334-fold decreased),and triglyceride levels were also declined significantly(0.1554-fold?0.3051-fold);Similarly,the different involvements in metabolic pathway between FMO-OE groups and the control groups mainly included amino acid and xenobiotics metabolism.More importantly,we also detected one compound with a quaternary amine structure(namely sinapine),whose contents were directly related to the expression levels of Dmfmos in vivo(15.26-fold?19.60-fold increased in FMO-KD groups and 11.93-fold?33.67-fold decreased in FMO-OE groups,respectively).However,whether it was a potential endogenous substrate of DmFMOs in Drosophila requiring further investigation.To sum up,this study focused on the metabolic characterization in male adult flies with Dmfmos knockdown or overexpression,which might provide valuable clues for the exploration of endogenous substrates of FMOs.?.Expression levels of Dmfmos could affect the life span and eclosion in D.melanogasterIn this study,we measured a range of the indexes related to the growth and development in transgenic fruit fly strains.Compared with the control group,downregulation of Dmfmos could reduce the Drosophila life span of fruit flies(p<0.0001),and Dmfmo1 had a more significant effect.On the contrary,overexpression of Dmfmos could prolong their longevity(p<0.0001),and Dmfmo2 had a more significant influence;the average weights of mid-aged fruit flies in FMOs-OE groups were between 0.74 mg and 0.77 mg,which were significantly decreased compared with the control group(0.79 mg).However,no significant differences were found between the FMOs-KD and control groups.Also there were no remarkable differences in metabolic rates and food consumptions between the transgenic strains of Drosophila and the controls.Additionally,since the number of offsprings with Dmfmo1 knockdown was abnormally reduced under the same feeding condition,then the eclosion rates were evaluated between the FMOs-KD and control groups,which showed that downregulation of Dmfmo1 could significantly cause the eclosion defect.These results indicated that expression levels of Dmfmos could affect the life span and the normal eclosion in fruit flies. |
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