Effect On Metabolic Health Of Offspring Exposure To Maternal Intrauterine Hyperlipidemia And The Mechanism Involved

Part Ⅰ Maternal hypertriglyceridemia induces hypertension in offspring by epigenetically reprogramming leptin levelsObjective:The present study was designed to investigate the effect of maternal hypertriglyceridemia (mTG) on blood pressure in offspring, and, to explore whether leptin is involved in reprogramming metabolic health in offspring when exposure to intrauterine hypertriglyceridemia.Materials and Methods:We first performed a clinical investigation in2525Han Chinese people, including1755children and770adults. BMI and blood pressure (BP) were measured in all subjects. We collected95blood samples for leptin analysis. The third trimester serum triglyceride levels were analyzed in2687Han Chinese women, including2375women who delivered singleton babies with normal birth weight (2500-3999g) and312women who delivered singleton macrosomic babies (≥4000g). Among these women, we recruited68umbilical cord blood samples and examined serum leptin levels. Human lymphocytes were collected to test the methylation of leptin using bisulfite squencing PCR. We then set a high fat diet pregnant rat model for further confirmation. The development of gestational hypertriglyceridemia was confirmed by testing the serum lipids during pregnancy. Offspring of dams underwent blood pressure testing by standard methods and orbital blood collection at3weeks,8weeks,6months and1year of age. Serum and lymphocytes were separatedly prepared for analysis of serum leptin levels, and, mRNA expression and methylation of leptin promoter. Visceral fat and subcutaneous fat were collected at birth and different ages of life for analysis of leptin mRNA levels and methylation rates. Leptin methylation was detected by the same methods.Results:The clinical investigation showed elevated serum leptin levels in individuals who were macrosomic at birth which were persistent from birth to adulthood, even though their BMIs had returned to normal by school age. Adults who were macrosomic at birth had increased blood pressures and incidence rates of hypertension. Meanwhile, the mothers of macrosima presented an obviously increased TG levels during prengnany, and the incidence of macrosomia increased significantly in high mTG group. Moreover, serum leptin levels were elevated in offspring of women with gestational hypertriglyceridemia, and they were positively correlated with maternal triglyceride levels, both in newborns and preschool age children. We then demonstrated that Leptin hypomethylation and up-regulation of TET1in human lymphocytes not only from umbilical blood but also from preschool age children. Using a high-fat-diet pregnant rat model, we confirmed that high maternal triglyceride levels permanently increased offspring serum leptin levels from birth to adulthood, and were associated with increasingly elevated blood pressure despite normal body weight. High maternal triglyceride levels induced leptin promoter hypomethylation and up-regulation of TET1were further confirmed both in adipose tissues and lymphocytes. Treatment of mesenchymal stem cells with free fatty acid during adipogenic differentiation also resulted in increased leptin and TET1expression as well as leptin promoter hypomethylation.Conclusions:These findings demonstrate that exposure to intrauterine hypertriglyceridemia can induce permanent elevations in serum leptin levels and hypertension in adulthood. Part Ⅱ High maternal hypertriglyceridemia may increase risk of hypercholesterolemia in offspring through modulation of leptin-mediated cholesterol metabolism by STAT3target gene CYP7alObjective:To investigate whether maternal hypertriglyceridemia (mTG) during gestation is associated with increased risk of hypercholesterolemia in offspring. To explore the mechanism that high leptin levels modulate cholesterol metabolism linking maternal hypertriglyceridemia to hypercholesterolemia in offspring.Materials and Methods:Follow-up study in children aged3-6years was performed.250mothers who delivered in our hospital were interviewed by phone call, and49children were willing to take part in this follow-up study in outpatient clinics. Maternal data of pregnancy was obtained via hospital records with patients consent. Blood samples were collected from those children and physical exams were performed including height, body weight and blood pressure. Hypercholesterolemia was defined according to the criteria of hyperlipidemia of National Cholesterol Education Program (NCEP, ATP, III). High fat diet pregnant rat model was set for further study. Blood samples and liver tissues were obtained from HFD offspring at3weeks old for analysis of leptin and lipid levels. Real-time RT-PCR and Western blot were used to examine the expression of key enzyme in cholesterol metabolism. Human hepatoma cell line (HepG2) was cultured for in vitro experiments. Different concentrations of leptin and (or) silence STAT3were used to treat cells, and quantitative PCR technique was used to detect expression of HMGCR, CYP7al and factors of LEPR-JAK2-STAT3passway; Chromatin immunoprecipitation was used to detect region of binding with STAT3in CYP7al promoter under leptin mediation.Results:Serum leptin and total cholesterol levels were elevated in offspring of women with gestational hypertriglyceridemia, and the rate of children hypercholesterolemia presented significantly higher in high mTG group. The total serum cholesterol levels were positively correlated with leptin levels in preschool age children. Using a high-fat- diet pregnant rat model, we confirmed that offspring born with high maternal triglyceride levels had a higher serum leptin and total cholesterol levels. The expression of CYP7al was obviously decreased in liver tissues of HFD offspring while there was no difference in expression of HMGCR. In vitro experiment showed that leptin can dose-dependently reduced CYP7al expression. Meanwhile, we found that the level of phosphorylated STAT3, an active form of STAT3, was dose-dependently increased when treated with leptin, and LEPR-JAK2was up-regulated as well. In order to clarify whether leptin down-regulated CYP7al via activating STAT3pathway, we transfected siRNA target STAT3into HepG2cells. Treatment with STAT3siRNA for48h significantly reduced the inhibitory effects of leptin on CYP7al expression. Chromatin immunoprecipitation analyses identified STAT3motif in the promoter of the CYP7al gene.Conclusions:Maternal hypertriglyceridemia is associated with high risk of hypercholesterolaemia in offspring. High leptin levels induced by maternal hypertriglyceridemia can modulate cholesterol metabolism by STAT3target gene cyp7al. Part Ⅲ Intergenerational transmission of abnormal lipid metabolism induced by high-fatdiet during pregnancyObjective:The study designed to determine the effect of intrauterine over-nutrition induced by high-fat diet (HFD) on lipid metabolism of F2offspring and how the effect transmitted to second generation.Materials and Methods:We fed pregnancy rats with FIFD and examined the lipid profiles of first generation (F1) and F2rats at different ages. We assessed the mRNA levels of imprinted genes in F1livers, F1sperms and F2livers. After adult liver tissues were collected, and real-time RT-PCR and bisulfite sequencing PCR were used to examine the expression and methylation of imprinted genes. Methylation status of the Igf2and Igf2r were analyzed and determined by cloning and sequencing of bisulfite-treated DNA.Results:The lipid profiles increased in3-week-,6-month-and1-year-old HFD-F1. Moreover, HFD-F2offspring presented elevated lipids from8weeks old till to6months old. The expression levels of Igf2and Igf2r were increased in HFD-F1and HFD-F2livers, which were further verified in HFD-F1sperms and HFD-F2fetal livers. In adult livers from HFD-F2rats, we found hypomethylated promoters of Igf2and Igf2r.Conclusions:HFD during pregnancy changes lipid metabolism of both F1and F2offspring. The modification of imprinted genes transmitted from F1male sperms to F2livers, thereby affects the health of offspring.