A Study Of The Impact Of Over-nutrient Environment During Prenatal And Postnatal Development To Glycolipid Metabolism In Female Offspring Mice

Background:In the past,it is generally accepted that chronic diseases in adults are caused by unhealthy lifestyle and environmental factors on the basis of genetic susceptibility of multiple genes.In 1995,David Barker and his colleagues put forward a new concept of the origin of the origin of human disease,Developmental Origins of Health and Disease(DOHaD).This hypothesis believes that the intrauterine environment is normal or not related to the offspring’s whole life and long-term disease.This hypothesis also illuminates that the environmental factors,especially nutrition factors play an important role in early life programming on metabolic disease and cardiovascular disease.High fat diet during prenatal(pre-pregnancy and pregnancy)and postpartum(lactation and early weaning)period plays an important role in the pathogenesis of metabolic syndrome.Numerous studies have shown that the diet and lifestyle during pregnancy are important determinants of maternal and offspring health.Too much energy and nutrient intake during pregnancy and lactation period is harmful,especially for overweight and obese women,abortion,gestational diabetes,preeclampsia,and high birth weight is increased,and incidence of obesity,type Ⅱdiabetes and cardiovascular disease of offsprings in adult stage is also increased.In addition,cancer is also associated with excess nutrition during pregnancy.Studies have shown that people with high birth weight are more likely to develop breast cancer and leukemia,and men with high birth weight have higher cancer incidence and mortality.A large number of studies are used to explain the pathogenesis of metabolic disease of offsprings caused by maternal high fat diet.It has been reported that high-fat diet led to not only obesity via the expanding of fat storage but also hyperglycemia which gradually resulted in notable insulin resistance.The patients suffering from T2DM were reported to exhibit progressively decreased number of pancreatic insulin-secreting β-cells as well as their function.Pancreatic β-cells produce insulin to facilitate the uptake of glucose.However,hyperglycemia gives rise to the overproduction of insulin from pancreatic β-cells to compensate for insulin resistance.Long-term hyperglycemia diminishes the sensitivity of insulin,resulting in glucose intolerance as well as probable pancreatic β-cell death,which eventually leads to diabetes.Elevated free fatty acids(FFAs)were proposed as another cause of insulin resistance correlated to diabetes that induces autophagosome accumulation in β-cells.It was demonstrated that maternal obesity or diabetes adversely reduced the pancreatic β-cell mass and therefore affected insulin secretion in their offspring.But,few studies have considered effects of over-nutrient environment during prenatal and postnatal(prepregnancy,pregnancy,lactation and early stage after wean)on offspring outcomes comprehensively.We will further study how over-nutrient environment act during prenatal and postnatal and how they interact with each other.In this research,we also chose microarray datasets from GEO and analyzed microarray data to provide theoretical base for the potential effect of prenatal HFFD to lipid metabolism.Methods:1.Female mice had ad libitum access to water and a standard normal chow(NC)diet.When indicated,mice were maintained on a high-fat/fructose diet(HFFD)and randomized to the water containing 10%fructose for 10 weeks before mating for 10 weeks before mating.The differences of body weight,fat content,blood glucose evaluation,plasma insulin level,glucose tolerance test,and insulin tolerance test between the two groups were compared.2.The female mice were mated with male mice fed on NC and tap water.Throughout pregnancy and lactation,female mice were maintained on their designated diets of either NC(n=10)or the HFFD(n=10).Before weaning,offspring in all litters were selected to only six female pups per damto achieve equal access to food,and the pups were randomly raised by female parents fed on either NC or HFFD,resulting in four individual groups,NC/NC(n=8),NC/HFFD(n=6),HFFD/NC(n=8),and HFFD/HFFD(n=6),in the order of prenatal/postnatal diet,respectively.After weaning at 3-weeks old,NC/NC female offspring still fed on NC for 10 weeks.NC/HFFD female offspring fed on HFFD for 10 weeks.HFFD/NC female offspring fed of NC for 10 weeks,and HFFD/HFFD female offspring fed on HFFD for 10 weeks.The differences of food intake,energy expenditure,body weight,plasma insulin level,blood glucose evaluation,plasma triglyceride level,Plasma free fatty acid level,plasma leptin level,insulin tolerance test,glucose tolerance test,fat content,liver weight,liver triglyceride content,histological analysis of white adipose tissue and liver tissue,morphological analysis of pancreatic beta cells,insulin release test,expression of proteins related to lipid metabolism in liver tissue,and expression of proteins and mRNA related to glucose metabolism in pancreas tissue were compared.3.We chose and downloaded 2 groups of microarray datasets of live of female offsprings fed by NC or HFFD during prenatal period from GEO.After noise reduction and normalization,2 groups of microarray datasets were combined in to a dataset.After the data pre-processed,we used unpaired t-test to screen differential genes.Finally,we used DAVID Software for GO analysis and KEGG pathway analysis,imported STRING online database for protein-protein interaction(PPI)network analysis,computed the network topology and got key genes through Cytoscape software.Results:1.From the appearance point of view,the HFFD group showed a more obvious trend of obesity at the 10th week of grouping(17thweek of birth).Body weight,fat content,blood glucose evaluation and plasma insulin level of HFFD were higher than NC obviously.Glucose tolerance test and insulin tolerance test showed obvious abnormality.2.Both NC/HFFD and HFFD/HFFD offspring exhibited obvious body weight and fat content gain,hyperglycemia,and severe insulin resistance.NC/HFFD and HFFD/HFFD offspring exhibited higher plasma leptin level compared to NC/NC and HFFD/NC mice.Meanwhile,HFFD/HFFD exhibited much higher plasma leptin level compared to NC/HFFD.When compared to NC/HFFD offspring,the HFFD/HFFD offspring exhibited more severe alterations in their metabolism and dysfunctions on pancreatic β-cells,suggesting a potential impact of prenatal HFFD on the programming of pancreatic β-cell deficiency in the fetus.NC/NC group and HFFD/NC group were almost normal.In NC/NC or HFFD/NC mice,phosphorylation of Akt at Serine 473 was markedly up-regulated in the presence of insulin,while reduced phosphorylated Akt protein expression was observed in NC/HFFD mice with insulin stimulation compared to NC/NC or HFFD/NC mice.Insulin stimulation was not able to induce Akt phosphorylation in HFFD/HFFD mice,which is another indicator of insulin resistance.Significantly higher protein and mRNA levels of insulin,PGC-1 a and G-6-Pase were found in NC/HFFD and HFFD/HFFD mice than NC/NC mice.Additionally,protein and mRNA expressions of GLUT-2 and GCK were both down-regulated in NC/HFFD and HFFD/HFFD mice compared to NC/NC mice.3.We discovered 85 differentially expressed genes in live of female offsprings fed by NC or HFFD during prenatal period,in which 38 are up-regulated and 47 are down-regulated.Cytoscape software screened key genes:LRP6,Prkd2,Kcnkl3 and Notchl.We also discovered Inositol phosphate metabolism,Wnt signaling pathway and Metabolic pathways associated with key genes.Conelusions:1.HFFD before pregnancy can successfully induce fat accumulation,insulin resistance,and hyperglycemia,which in turn elevated the risk of predispositionto obesity and T2DM based on previous study.There were overt relations between obesity and diabetes.2.The direct consequences of prenatal and postnatal HFFD were fat deposition,hyperglycemia,hyperinsulinemia,and β-cell dysfunction,which may lead to obesity and T2DM in the offspring.3 Leptin resistance and the functions of pancreatic β-cells changed were explanations for insulin resistance.4.Reduced phosphorylated Akt expression,increased insulin,PGC-la and G-6-Pase expressions,and decreased GLUT-2 and GCK expressions were important causes of abnormal glucose metabolism in offspring female mice.5.The metabolic dysfunction is more likely to arise from postnatal HFFD.Although prenatal HFFD cannot cause metabolic dysfunction by itself,but it is capable of causing adverse influence such as epigenetic alteration in the offspring.6.LRP6,.Prkd2,Kcnk13 and Notchl is a key gene of differentially expressed genes in live of female offsprings fed by NC or HFFD during prenatal period.Inositol phosphate metabolism,Wnt signaling pathway and Metabolic pathways were important pathways that associated with key genes.LRP6 plays an important role in the uptake and clearance of LDL,and LRP6 plays an important role in the formation and accumulation of liver fat.