Effect Of Inulin On Glycolipid Metabolism And Pregnancy Outcomes In Gestational Diabetes Mellitus

Incidence of Gestational diabetes mellitus（GDM）is world-widely increasing and leading to increased economic costs of overall healthcare significantly.Meanwhile,treatmentoptions for GDM are limited.In recent years,the development of micro-ecological technology provides a new method for the further research on gut microbiota.Gut microbiota will be a new target for the treatment of GDM.Inulin is known as a prebiotic which will not be digested and decomposed in the oral cavity and small intestinebecause of its specialβ-（2,1）-glycoside bond structure.Some studies have confirmed that inulin can reduce the levels of blood glucose and lipid,preventcancer,regulate the balance of intestinal flora,promote absorption of minerals and vitamins and relieve constipation.However,there are few reports on the effect of inulin on GDM.This study aimed to determine the effects of inulin on glucolipid metabolism and pregnancy outcomes in GDM.Meanwhile,C57BL/6J mice fed with high-fat/sucrose diet were used to reveal the mechanism.Part 1:Establishment of a mouse model of gestational diabetes mellitusObjective:Compare the formation and stability of animal model of gestational diabetes mellitusinduced by high-fat/sucrose diet（HFD）or streptozotocin.Methods:The body weights andoral glucose tolerance test（OGTT）of mice were measured during pregnancy.Blood sample were collected to analyze the fasting blood glucose（FBG）and insulin（FINS）concentrations.Maternal fetuses were weighted and measured on gestational age of 18 day（GD 18）.Results:（1）HFD1 group was fed with high-fat/sucrose diet after conception.The body weight of HFD1 group was significantly higher than NCD group at the middle and late stages of pregnancy;FBG of HFD1was also increased while the difference was not statistically significant;FINS and HOMA-IR of HFD1 on GD 18 were significantly higher than NCD group;All these showed that obesity and mild insulin resistance could be induced by high-fat/sucrose diet in the short term,but no hyperglycemia was found in HFD1 group.HFD2 group was fed with high-fat/sucrose diet 4 weeks before conception.The FBG on GD10,OGTT on GD14,FBG,FINS and HOMA-IR on GD18 of HFD2 group were significantly higher than those of NCD group.The weight,water consumption and urine volume of HFD2 group also increased significantly compared with NCD group.（2）The insulin secretion reduced significantly and the blood glucose increased rapidly in STZ60 mg/kg group.High rates of abortion and mortality were found in STZ60 mg/kg group due to the excessive and rapid increase of FBG.The state of hyperglycemia retained short time and unsteadily in STZ30 mg/kg group.The hyperglycemia of pregnant mice in STZ45mg/kg group lasted longer and more stable than the mice in STZ30 mg/kg group.The incidences of abortion and mortality of STZ45 mg/kg group were significantly reduced compared with STZ60 mg/kg group.The optimum dose to construct ideal experimental animal model of STZ-induced GDM was STZ 45 mg/kg.However,the FINS and HOMA-IR of GD18 in STZ 45 mg/kg group were lower than those in NCD group which were not consistent with the GDM patients.Therefore,the animal model of GDM induced by STZ was not selected to establish the experimental animal model for the further study.Conclusion:The C57BL/6J mice fed with high-fat/sucrose diet 4 weeks before conceptionand throughout gestation is a good method to establish a mouse model of gestational diabetes.Part 2 Effects of inulin on glycolipid metabolism and pregnancy outcomes in gestational diabetic mice and its underlying mechanismObjective:Observe the effects of inulin on glycolipid metabolism and pregnancy outcomes in GDM mice and analyze the possible molecular mechanism.Methods:C57BL/6J mice were fed with HFD4 weeks before conception and throughout gestation to mimic chronic maternal fast food consumption.Based on this GDM mouse model,inulin was given to GDM mice by gavage.Female C57BL/6J mice were divided into 4groups randomly:normal control group（NCD）,gestational diabetes metabolism group（GDM）and two different dose inulin（3.33g/kg.d and 1.67g/kg.d）groups.During pregnancy,the body weight was measured everyday and the OGTT were performed on GD14.The blood was collected on GD 18 to analyze the FBG,FINS,triglyceride（TG）,total cholesterol（TC）,low-density lipoprotein（LDL）and high-density lipoprotein（HDL）concentrations.HE staining was used to checkthe steatosis of hepatic and adipose tissue.RT² profiler PCR Arrays were performed to screen differentially expressed genes which were associated with glycolipid metabolism.These genes were verified by Real-time PCR assay in the liver,fat and placenta tissues of mice.Western blotting was used to detect the expressions of proteins related to the most differentially expressed gene in the glycolipid metabolism pathway.Results:（1）Inulin can significantly reduce the body weight of mice in GDM group.The results of serum biochemical index of mice showed that FBG,TG,TC and LDL levels in GDM group were higher than those in NCD group.Inulin-treated mice had decreased FBG,TG,TC and LDL levels compared with GDM group（p<0.05）.The AUC and HOMA-IR in inulin-treated groups were significantly lower than the GDM group.There was a certain concentration-response relationship between the two inulin-treated groups.（2）There were 12 expressed differentially genes screened out by RT² profiler PCR Arrays:G6pc,Slc2a4,Pdhb,Pgk1,Pgm3,Pdk2,Pfkl,Igfbp5,Retn,Pdk4,Prps1and Phka1.Western blotting was used to detect the expressions of insulin receptor substrates（IRS）,protein kinase B（Akt）and extracellular regulated protein kinase（ERK1/2）in the liver and adipose tissues related to the Retn（Resistin）which was the most differentially expressed gene verified by Real-time PCR assay.Conclusion:Inulin can improve maternal glucose and lipid metabolism in GDM mice through Resistin-IRS and Resistin-Akt signalling.Part 3 Effects of inulin on the intestinal microbiota in gestational diabetic miceObjective:Study the effects of inulin on the abundance and diversity of intestinal microbiota and explore the characteristic intestinal microbiota species related to the disturbance of glycolipid metabolismin GDM mice.Methods:Ion S5^TMXL sequencing platforms were used to detect the V4 and V5 hypervariable 16S r RNA regionto explorethe difference of abundance and diversity of intestinal microbiota among gestational diabeticmice,normal mice and inulin-treated mice.Results:The ratio of Firmicutes to Bacteroides increased significantly in GDM group.The decrease of the relative abundance of butyrate-producing bacteria（Bacteroidales group and Ruminococcaceae families）and the increase of the relative abundance ofgram negative conditional pathogen（Erysipelotricchia）were found in GDM group.Inulin could increase the relative abundance of butyrate producing bacteriaand reduce the number of opportunistic pathogens.The relative abundance of Akkermansia also increased significantly after inulin intervention.Conclusions:Inulin could increase the abundance and species diversity of intestinal microbiota,promote the relative abundance of probiotics in intestine significantly,reduce the number of conditional pathogens and improve the structure of intestinal flora in GDM mice.Inulin is potentially effective functional food for the prevention and/or treatment of GDM.