| Background and PurposeThe prevalence of diabetes in young people has increased over the recent years due to obesity,lack of exercise and western diet.Two in five women with diabetes are at childbearing age and one in seven pregnant women suffer from Gestational diabetes mellitus(GDM).Pregnant women with Gestational diabetes mellitus are often obese.Gestational diabetes mellitus will not only increase the risk of type 2 diabetes mellitus(T2DM)in women with GDM pregnancy,but also cause pregnancy complications such as fetal malformations,hyperinsulinemia,and respiratory distress syndrome of newborn.Moreover,offspring of pregnant women with GDM have a significantly increased risk of obesity and impaired glucose metabolism in the future.Mothers’ high-fat diet lead to impaired glucose metabolism during pregnancy and/or lactation,which can increase the risk of type 2 diabetes in offspring.However,the underlying causes for this epidemic condition remain elusive.We propose that maternal transmission of gut microbiota plays a major role in the onset of diabetes in the young,if proven,will lead to a viable approach for the prevention.Studies have shown that original gut microbiota of infants might be seeded before birth and are mainly affected by the way of delivery and lactation.Specifically,caesarean section(CS)has long-lasting effects on gut microbiota composition in conventional mice.Besides,cross-fostering immediately after birth induces a permanent microbiota shift that is shaped by the nursing mother.Therefore,this study tested the hypothesis that the maternal gut microbiota plays a key role in regulating glucose homeostasis in offspring through gut-islet axis and investigate the effects of intrauterine environment,delivery mode,and lactation way on transmission of gut microbiota.Methods1.Animals,breeding,and CSFemale C57BL/6J mice at 7 weeks were placed either on a high fat diet(HFD,containing 60%fat and a total of 5.24 kcal/g)or a normal chow diet(NCD,containing 11.4%fat and a total of 3.90 kcal/g)ad libitum.After 8 weeks of dietary intervention,the two groups of female mice were mated with mature normal male C57BL/6J mice.Caesarean section was conducted at day 20 of pregnancy by hysterectomy,and pups were handled aseptically before they were transferred to a foster mom.Cross-fostering was then performed shortly after birth.Nomenclature used for pups born by caesarean section or vaginal delivery and for natural or adoptive mothers are as follows:normal-chow diet group;high-fat diet group;high-fat diet with CS group;normal-chow diet with CS group;high-fat diet with cross-fostering group;normal-chow diet with cross-fostering group;high-fat diet with CS and cross-fostering group;normal-chow diet with CS and cross-fostering group.Excluding individuals with extremely low and high weight,10 offspring from each group were selected.At weaning(postnatal day 21),all offspring were placed on normal chow diet until 8 wk of age.2.Measurement of body weight,glucose tolerance and insulin tolerance testsBody weight of mothers were monitored weekly during the first trimester,gestation,and lactation.The offspring’s body weight was determined at born,3 to 8 wk of age.Oral glucose tolerance test was performed by gavage in offspring at 4 and 8 wk of age,and the blood glucose were measured at each time point.Insulin tolerance tests were performed subsequently.3.Islet insulin secretory response and pancreatic transcription factor in pancreatic tissueAt 8 wk,pancreatic islets were isolated and collected individually using a microscope.In vitro insulin release was then measured with a basal glucose concentration(3.3mmol/L)and high glucose concentration(16.7mmol/L).Pancreatic tissues were dissected at 8 wk for mRNA isolation,and the mRNA expression levels of pancreatic specific transcription factors Pdx-1,MafA genes were detected by quantitative PCR.4.16S rRNA gene V3-V4 region sequencing and data analysisFecal samples were collected from dams and offspring at 4 and 8 wk in each group,and 16S rRNA gene V3-V4 region sequencing was performed on the Illumina MiSeq platform.The statistical significance between different groups was analyzed with multivariate analysis of variance.5.SPSS 24.0 statistical software for data analysis.Results1.Maternal high-fat diet caused significant increase in fasting blood glucose and fasting insulin(P<0.05),and led to impaired glucose tolerance(P<0.05).2.The weight of offspring of high-fat diet group was significantly higher than that of normal chow diet group(P<0.05).Glucose tolerance test showed that blood glucose-levels of offspring of high-fat diet group were higher than those of the normal chow diet group at each time point(P<0.05).It also showed impaired insulin tolerance in offspring of high-fat diet group(P<0.05).Insulin secretion of isolated islets at high glucose state was decreased(P<0.05).Pancreatic specific transcription factor Pdx-1 and MafA mRNA expression decreased(P<0.05).Sequences of 16S rRNA gene V3-V4 region showed significant differences in the structure of gut microbiota in offspring between high-fat diet group and normal chow diet group,which was similar to their mothers’ flora.3.After caesarean section,compared with offspring of high-fat diet group,blood glucose levels of offspring from high-fat diet with CS group were unchanged.Its impaired insulin tolerance was alleviated(P<0.05).Insulin secretion of isolated islets at high glucose state was partly recovered(P<0.05).Pancreatic specific transcription factor Pdx-1,MafA mRNA expression levels were slightly increased.Sequences of 16S rRNA gene V3-V4 region showed significant differences in the structure of gut microbiota of offspring between the two groups.Compared with offspring of normal-chow diet group,there was no significant change in blood glucose and serum insulin levels of offspring from normal-chow diet with CS group,while it showed a slight insulin resistence(P<0.05).The isolated pancreatic islet function was not significantly improved.The mRNA expression of Pdx-1 in the pancreas was slightly decreased,but there was no statistical significance.At the age of 4wk,the structure of gut microbiota of this group was different from that without CS,but the difference was not significant at the age of 8wk.4.After cross-fostering,compared with offspring of high-fat diet group,blood glucose levels of offspring from high-fat diet with cross-fostering were reduced(P<0.05),and impaired insulin tolerance was alleviated(P<0.05).Insulin secretion of isolated islets at high glucose state was significantly recovered(P<0.05).Pancreatic specific transcription factor Pdx-1 and MafA mRNA expression levels were partly increased(P<0.05).Sequences of 16S rRNA gene V3-V4 region showed significant differences in the structure of gut microbiota in offspring between the two groups.Compared with offspring of normal chow diet group,there was a slight increase in blood glucose levels of offspring from normal chow diet with cross-fostering group(P<0.05),as well as a slight insulin resistence(P<0.05).The isolated pancreatic islet function was slightly improved and the mRNA expression of Pdx-1 in the pancreas was slightly decreased,but there was no statistical significance.It also showed significant differences in the structure of gut microbiota in offspring between the two groups.5.After caesarean section and cross-fostering,compared with offspring of high-fat diet group,blood glucose levels were further reduced in offspring from high-fat diet with both caesarean section and cross-fostering(P<0.05).Impaired insulin tolerance was alleviated(P<0.05).Insulin secretion of isolated islets at high glucose state was significantly recovered(P<0.05).Pancreatic specific transcription factor Pdx-1 and MafA mRNA expression levels were increased(P<0.05).Sequences of 16S rRNA gene V3-V4 region showed significant differences in the structure of gut microbiota in offspring between the two groups,and the structure of gut microbiota in offspring from high-fat diet with both caesarean section and cross-fostering was closer to that in normal-chow diet group.Compared with offspring of normal-chow diet group,there was a significantly increase in blood glucose levels of offspring from normal chow diet with both caesarean section and cross-fostering group(P<0.05),as well as insulin resistence(P<0.05).The isolated pancreatic islet function and the mRNA expression of Pdx-1 in the pancreas were slightly decreased(P<0.05).It also showed significant differences in the structure of gut microbiota in offspring between the two groups,and the structure of gut microbiota in offspring from normal chow diet with both caesarean section and cross-fostering was closer to that in high-fat diet group.6.Cesarean section combined with cross-fostering had the greatest impact on the gut microbiota of offspring,followed by cross-fostering only.There are 5 CAGs represented by Bifidobacterium,,Lactobacillus murinus,and Muribaculaceae which,showed significant differences in offspring of high-fat diet and normal chow diet group at 4 wk.After cross-fostering,the abundance of these CAGs has changed significantly.Meanwhile,CAG3(represented by Bifidobacterium)was significantly higher in offspring of high-fat diet group than normal chow diet group at both 4 wk and 8 wk.Simultaneously,both caesarean section and cross-fostering significantly reduced the abundance of CAG3 in offspring of chow diet group.ConclusionBoth caesarean section and cross-fostering have profound effcets on glucose metabolism and islet function of offspring from high-fat diet dams,and blocking the vertical transmission of dysfunctional gut microbiota via delivery mode and lactation way is the key pathway to repair disruption of gut microbiota on remittence of metabolic disorders efficiently. |
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