| Part 1Maternal exercise and genistein intake improve the deleterious effects of early-life overnutrition on glucose and lipid metabolism in offspring Objectives:The global prevalence of diabetes is increasing rapidly,but the pathogenesis is not yet fully understood.The health resources of diabetes and lipid metabolism disorders in China are mostly distributed after the disease has developed or after the complications occur,thus the early identification and intervention of the disease is urgent.In recent years,more and more studies have shown that early-life malnutrition significantly increases the risk of diabetes and obesity in adulthood.This study aims to explore whether maternal exercise and genistein intervention can improve or even reverse the abnormal metabolism of offspring in adulthood caused by early-life overnutrition via animal models.Materials and Methods:Two independent mouse cohorts were generated to verify thehypothesis that "maternal exercise and genistein intervention can effectively improve glucose and lipid metabolism in adult offspring":1.maternal exercise mouse model:C57BL/6J mice were randomly divided into 3 groups according to their body weight,including a control diet group,a high-fat diet group,and a high-fat diet and exercise group,which was given a voluntary wheel running.The exercise intervention was 3 weeks before pregnancy and during pregnancy,and the diet intervention was 3 weeks before pregnancy and through pregnancy and lactation.2.maternal genistein intervention mouse model:C57BL/6J mice were randomly divided into 3 groups,including a control diet group,a high-fat diet group,and a high-fat diet with genistein group(0.6g/kg body weight)3 weeks before pregnancy and during pregnancy and lactation.In the two mouse models,female mice were mated with C57BL/6J males of the same age with control diet.After weaning,the offspring were fed a normal control diet to 24 weeks of age.We measured body weight,birth weight,fat mass,food intake,exercise distance and glucose tolerance test of the dams and offspring.The dams at weaning and the 24-week-old offspring were sacrificed to collect peripheral blood,which was used to detect insulin and blood lipid profiles to analyze the effects of maternal exercise and genistein intervention on metabolism in adult offspring.Results:1.The high-fat diet 3 weeks pre-pregnancy and during pregnancy and lactation significantly increased the blood glucose,fasting serum insulin,serum total cholesterol and low-density lipoprotein cholesterol levels of dams than that of the control diet group.However,maternal exercise has limited effects on glucose and lipid metabolism,and only tended to reduce the insulin resistance index HOMA-IR in dams.Nevertheless,the maternal high-fat diet caused catch-up growth and a tendency to become obese with age in offspring.At the same time,the area under the glucose tolerance test curve,the subcutaneous and visceral adipose tissue mass increased significantly at the age of 24 weeks.Insulin resistance,hypercholesterolemia and hypertriglyceridemia also appeared.Maternal exercise can prevent obesity,reduce blood glucose and fat mass,and improve insulin resistance and dyslipidemia induced by maternal high-fat diet in adult offspring.2.In the maternal genistein intervention mouse model,we also found that the maternal high-fat diet caused significant impaired glucose metabolism and dyslipidemia in the dams and offspring,while genistein intake tended to decrease the fasting serum insulin levels and significantly reduced fasting blood glucose levels during pregnancy in dams.For offspring,maternal dietary genistein supplementation can prevent catch-up growth after birth,significantly reduce the area under the glucose tolerance test curve,the subcutaneous and visceral fat mass,and improve insulin resistance and dyslipidemia at 24 weeks of age in offspring.Conclusions:Maternal voluntary wheel running during the 3 weeks pre-pregnancy and pregnancy can significantly improve the offspring's susceptibility to obesity,and disorders of glucose and lipid metabolism caused by high-fat diet early in life;maternal genistein intake 3 weeks before pregnancy and through gestation and lactation also reduced the body fat mass,and improved the abnormal glucose metabolism,insulin resistance and blood lipid profile disorders of adult offspring induced by early-life high-fat diet.Therefore,maternal exercise and dietary genistein intervention can effectively improve or even reverse the poor "metabolic memory" induced by ovrnnutrition,and timely prevent the occurrence of abnormal glucose and lipid metabolism in adult offspring,which made preventing the occurrence and development of metabolic abnormalities in early life become possible.Part 2The role of gut microbiota in mediating the effects of maternal intervention on glucose and lipid metabolism in adult offspringObjectives:The gut microbiota plays an important role in metabolic health.In recent years,emerging studies have suggested that the intestinal microbiota may play an important role in mediating the effects of adverse developmental environment in early life on metabolic abnormalities in adulthood,thus it might be one of the key mechanisms underlying the"metabolic memory".Given that both exercise and dietary genistein can regulate the gut microbiome,we aim to explore whether the intestinal microbiome plays an important role in deciphering the beneficial effects of maternal exercise and genistein intervention on metabolism in adult offspring.Materials and Methods:Based on the maternal exercise and dietary genistein intervention animal model established in the first part,the cecal contents of the dams at weaning and 24-week-old offspring were obtained.The gut microbiome 16s rRNA amplicon sequencing was performed using Illumina Hiseq 2500 PE250.Bioinformatic analyses including OTU analysis,species annotation,species difference analysis,and diversity analysis were done.We also correlated the significantly different species and metabolic pathways.Spearman correlation analysis was performed to correlate the significantly altered genera and metabolic parameters.Results:1.3 diversity analysis showed that the gut microbiota of mothers was significantly separated among the control diet group,high-fat diet group and high-fat diet and exercise group.At the same time,exercise significantly increased the abundance of Alloprevotella,Barnesiella,Odoribacter and Saccharibacteria_genera_incertae_sedis in the intestines of dams.For adult offspring,? diversity analysis showed that the gut microbiota was also significantly separated among the three groups.Maternal high-fat diet significantly enriched the Lachnospiracea_incertae_sedis and Anaeroplasma at the genus level,while maternal exercise significantly increased the abundance of Helicobacter,Odoribacter and Clostridium XIVb in the adult offspring.Spearman correlation analysis showed positive correlation between the abundance of Anaeroplasma and the blood glucose and area under the curve during the glucose tolerance test,fasting insulin,HOMA-IR index,and total cholesterol levels.Lachnospiracea_Incertae_sedis was also positively correlated with blood glucose and free fatty acid levels.In contrast,the Helicobacter and Odoribacter increased by maternal exercise were significantly negatively correlated with the blood glucose,insulin and lipids of adult offspring.2.In the dietary genistein intervention mouse cohort,? diversity analysis showed that the maternal gut microbiota among three groups was significantly separated.The high-fat diet significantly enriched Mucispirillum,which was reversed after genistein intervention in dams.The abundance of Barnesiella,Clostridium XIVa,Clostridium IV,Eubacterium and Bifidobacterium was significantly lower in high-fat diet group than that of the control group,among which Allobaculum,Barnesiella,Clostridium XIVa and Eubacterium were rescued by genistein.For adult offspring,? diversity analysis showed that intestinal microbiota was significantly separated after the intervention of maternal high-fat diet and genistein supplementation.At the genus level,maternal high-fat diet significantly reduced the abundance of Bifidobacteria in offspring.Erysipelotrichaceae_incertae_sedis was significantly enriched in offspring of maternal genistein intake group.Meanwhile,Erysipelotrichaceae_incertae_sedis and Bifidobacterium were negatively correlated with serum fasting insulin,lipids and white fat mass in adult offspring.Conclusions:1.Maternal high-fat diet and exercise significantly separated the intestinal microbiota of dams and adult offspring,and their composition and structure were also changed.The altered bacteria were significantly correlated with metabolic parameters.Among them,the reduction in harmful bacteria Lachnospiracea_incertae_sedis and the significant enrichment of short-chain fatty acid-producing bacteria may be the key factors mediating the intergenerational metabolic regulation of maternal exercise.2.Maternal dietary genistein intake also regulated the gut microbiota of dams and offspring,which was correlated with the improved metabolic parameters.Among them,the intestinal short-chain fatty acid-producing bacteria and Erysipelotrichaceae_incertae_sedis were significantly enriched,but the harmful species Mucispirillum was reduced by maternal genistein intake,which might mediate the intergenerational metabolic benefits.In addition,Bifidobacterium was associated with improved metabolism of dams and offspring.This study originally explored the role of gut microbiota in mediating the intergenerational metabolic effects of exercise and genistein intake,which might provide some theoretical evidence and potential intervention targets in early life for preventing the occurrence and development of adulthood metabolic abnormalities.Part 3Epigenetics might be a crucial mechanism underlying the improvement of metabolic health in adult offspring by maternal exercise and genistein interventionObjectives:Epigenetics mediates the interaction between genes and the environment,and plays an important role in metabolic regulation.With the rapid development of high-throughput sequencing technology,recent evidence shows that miRNAs may be one of the key molecules that mediate the metabolic programming effects of early-life malnutrition.However,the research exploring the effects of maternal exercise and dietary genistein intake on epigenetic modification in adult offspring is limited.This study aims to explore the programming effects of maternal exercise and genistein intake on liver metabolism in adult offspring and whether miRNAs play an important regulatory role in it.Materials and Methods:Based on the animal model of maternal exercise and dietary genistein intervention established in the first part,we obtained the liver tissues of adult offspring at 24 weeks of age,and then performed RNA extraction,transcriptomics,and miRNAs sequencing,followed by bioinformatics analysis.Finally,the qRT-PCR was used to verify the sequencing results.Results:1.Compared with the control group,maternal high-fat diet resulted in a significant upregulation of 195 genes in the liver of adult offspring,and a downregulation of 158,while maternal exercise significantly up-regulated 285 genes and down-regulated 324 genes compared with that of high-fat diet group,among which the expression of 122 genes was significantly increased by maternal high-fat diet and was decreased by exercise.KEGG pathway analysis showed that the 122 genes were mainly enriched in signaling pathways related to glucose and amino acid metabolism;while 89 genes were significantly down-regulated by maternal high-fat diet but were up-regulated by exercise,which were mainly enriched in unsaturated fatty acid biosynthesis,PPAR signaling pathway and fatty acid metabolism pathway.2.Compared with the control group,there were 20 different miRNAs in the offspring liver of maternal high-fat diet group;maternal exercise significantly up-regulated 7 miRNAs,while down-regulated 13 miRNAs compared with that of the high-fat diet group.Among them,3 miRNAs,namely miR-204-5p,miR-10b-5p and miR-139-3p,were significantly decreased in high-fat diet group,while exercise increased their expression.Their target genes were involved in epigenetic regulation and cholesterol biosynthesis regulated by SREBF pathway.3.Compared with control group,the differentially expressed genes in adult offspring caused by maternal high-fat diet were significantly enriched in sterol hormone biosynthesis,primary bile acid biosynthesis,TGF? signaling pathway.and diabetic complications related pathways.Maternal genistein intake significantly regulated 49 genes compared with high-fat diet group,of which 32 were up-regulated and 17 were down-regulated.These genes were significantly enriched in pathways related to glucose and amino acid metabolism.10 genes were reversed by maternal genistein intake,which were mainly enriched in pathways related to immune regulation and inflammation.4.Compared with the control group,there were 20 different miRNAs in the offspring liver of maternal high-fat diet;maternal dietary genistein intake changed the level of 27 miRNAs compared with high-fat diet group,of which 10 miRNAs were up-regulated,while 17 miRNAs were down-regulated.3 miRNAs,namely miR-12206-5p,miR-200a-5p and miR-21a-3p,were significantly decreased in high-fat diet group,and were reversed by maternal genistein intake.Their target genes were predicted to be involved in pathways associated with glucose,lipid and amino acid metabolism.Conclusions:1.Maternal exercise significantly regulated liver metabolism in adult offspring at the molecular level,and reversed the adverse effects of maternal high-fat diet on offspring.The reversed genes were enriched in glucose and lipid metabolism related pathways.At the same time,it can significantly rescue the changes in the expression levels of 3 miRNAs in livers of adult offspring,and their target genes were involved in the regulation of cholesterol biosynthesis and epigenetic modification,which may play an important role in mediating the intergenerational metabolic regulation of exercise.2.Maternal genistein intake not only improved phenotypes,but also regulated hepatic metabolism in adult offspring at the molecular level.Genes that were reversed by maternal genistein were significantly enriched in immune regulation-related pathways.Additionally,maternal genistein intake up-regulated the reduced miRNAs caused by maternal high-fat diet in adult offspring livers,including miR-12206-5p,miR-200a-5p,miR-21a-3p,and these miRNAs were predicted to be involved in the regulation of glucose and lipid metabolism and inflammatory response,which may be one of the key molecules that mediate the intergenerational effects of dietary genistein intake.In conclusion,this study pioneered the role of epigenetic regulation in mediating the programming effects of maternal exercise and genistein intervention on adult offspring metabolism,providing novel insight into the reversal of "metabolic memory". |
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