The Mechanism Of Reducing Blood Sugar And Lipid By DNJ From Mulberry Leaves And Screening Of DNJ Producing Strains

Mulberry leaf is one of the important resources of medicine and food with multifaceted functions,such as regulation of blood glucose,blood fat and harbors anti-aging,anti-tumor,anti-inflammation attributes.1-deoxynojirimycin(DNJ)is a kind of alkaloid obtained mainly from mulberry and has been involved in lowering of blood glucose,blood fat and delaying aging.On the basis of previous studies,this work was executed with type II diabetes db/db mice model and investigation of biochemical parameters such as blood glucose,insulin,blood lipids and other biochemical indicators alterations after the DNJ administration.Subsequently,we have used the proteomics methods to explore the hypoglycemic mechanism of DNJ from two levels of transcription and metabolism.Further,mulberry rhizosphere soil was screened for DNJ producing bacterial strains and their identification.In this paper,the mechanism of DNJ regulation of blood glucose and blood lipid in mulberry leaves was studied,which provided a theoretical basis and guidance for the development of mulberry leaves for function food,and established the foundation for the development of new sources of DNJ.We have evaluated the effects of DNJ extracted from mulberry leaf on the body index of db/db mice.Compared with the db/db mice without intervention,the body weight and blood biochemical parameters of db/db mice with type II diabetes mellitus were significantly changed after 9 weeks of DNJ supplementation with mulberry leaves.Firstly,DNJ showed the controlled effect on the weight of db/db mice and significantly decreased the content of blood glucose and insulin.The blood glucose levels decreased from 32.6 mM to 27.6 mM;insulin content decreased from 67.6 mIU/L to 27.1 m IU/L and insulin resistance was reduced from 97.87 to 33.23,which showed that DNJ has hypoglycemic effect and could lead to increased insulin sensitivity.Secondly,DNJ had significant influence on the content of serum lipids: TC decreased from 4.16 mM to 3.08 mM;TG decreased from 2.36 mM to 1.34 mM;LDL decreased from 3.54 mM to 3.49 mM;and the high quality protein HDL increased from 1.06 mM to 1.20 mM,demonstrating the remarkable effect of DNJ in db/db mice blood lipid profile.Further,DNJ could significantly reduce the content of MDA in the serum of db/db mice from 26.17 nM to 18.10 nM,indicating increased antioxidant ability.At the same time,AGEs also showed a downward trend,reducing the incidence of diabetic complications.Finally,DNJ considerably decreased the content of Cr in serum from 90.71 M to 61.17 M and the contents of ALT and BUN also decreased.The liver cells staining observed in DNJ intervention group revealed the protective effect of DNJ on liver and kidney function and maintaining the integrity of liver cells in the untreated group.We have analyzed the hypoglycemic mechanism of DNJ extracted from mulberry leaf through liver transcriptome.The differences of DNJ gene expression in db/db mice(DMT group)and wet pre group(DM group)were compared.According to the results of transcriptome sequencing,total 2024 differentially expressed genes were screened out,of which up to 398 were up-regulated and the remaining 1626 were down-regulated.Through GO analysis,it was observed that differentially expressed genes were responsible for carbohydrate metabolism,lipid metabolism,circulatory system,immune system,transferase activity,dehydrogenase activity and secondary metabolism processes;through KEGG analysis,the genes for 42 signaling pathways and 217 metabolic pathways were observed which were mainly involved in carbon and lipid metabolism pathway,energy metabolism and amino acid metabolism pathway,inorganic salt transport and signal transduction pathway.Subsequently,after the results obtained from the transcriptome,the genes responsible for metabolism of glucose and lipid were analyzed by quantitative PCR validation.Firstly in the sugar metabolism,DNJ could significantly reduce the expression of G6 PC and PEPCK mRNA,whereas up-regulate effectively to promote the HK mRNA expression.Our results also showed that DNJ could promote glycolysis.Secondly,in case of lipid metabolism,DNJ can considerably reduce lipid metabolism related genes such as SCD,CYP4 A,PAP,ATPase,ACADVL,APOA4 mRNA,which resulted in reduced lipid formation,oxidation of fatty acids,cholesterol synthesis as well as fat accumulation.Finally,DNJ significantly decreased the expression of CAMK2,ATP1A(mainly expressed in insulin secretion pathway),reduced the insulin secretion and resulted into decreased insulin resistance.In addition,DNJ treatment could significantly reduce the expression of Apolipoprotein A-IV,UGT,DPYD,CPS1,AADAT,ALDH6A1 and ADCY genes.These results suggested that DNJ could reduce blood glucose and blood lipid through multiple pathways.We have analyzed the hypoglycemic and hypolipidemic mechanisms of DNJ extracted from mulberry leaf through urinary metabolic profile.Diabetic mice group(DM)was targeted to compare the urine metabolism with DMT group and non intervention group with LC-MS.Total 38 metabolites were significantly altered between cis-and di-acetaldehyde.Regarding the glucose and lipid metabolism,the differences were analyzed and it was found that DNJ can significantly reduce the content of 3-glyceraldehyde-3-phosphate,galactitol,CIS aconitic acid in the urine.DNJ can promote the glucose into the tricarboxylic acid cycle.DNJ significantly reduced the content of palmitoyl carnitine lombricine,2-methoxyestrone 3-sulfate,acetylacetic in urine and led to decreased production of lipids and ketone bodies.In addition,DNJ changed the content of amino acid metabolites in the urine which noticeably improved the content of piperidine acid and significantly reduced the argininosuccinate,phenylethylamine,benzeneacetaldehyde,acid,alkali,guanidyl valeric acid and phenol content.All these changes showed that DNJ has a regulatory role on amino acid metabolism.We screened and identified the DNJ producing strains and optimized the DNJ fermentation.A glycosidase inhibitor producing strain was obtained by using the method of glycosidase inhibition and screening.The inhibitors were identified by combining mass spectrometry and nuclear magnetic resonance,and further identified as DNJ.Through morphological,physiological and biochemical characterization and 16 S rDNA identification,the strain was identified as Bacillus W642 and named as Bacillus pumilus.Through the single factor experiment,the optimize fermentation conditions obtained for B.pumilus W642 DNJ were as followed;the optimum medium consisted of 4% KNO3,5% beef extract,2% sorbitol,0.5% MgSO4 at pH 7 and 30 ? with 150 RPM shaking for 3 days.Finally,the highest yield achieved in the fermentation liquid of DNJ was up to 210 mg/L,which was observed to be 50% higher than under normal conditions.