| Early epidemiological investigations indicate that there is an increased susceptibility to metabolic syndrome in fetus with intrauterine growth retardation (IUGR), and the susceptibility has the characteristic of atavism, which suggests that there is an intrauterine origin of metabolic syndrome. However, the mechanism is still unknown. Previous researches show that smoking during pregnancy can lead to IUGR, and the risk of developing metabolic diseases is also increased in later adulthood life. Nicotine is the main component of tobacco. Our recent studies show that prenatal xenobiotics (such as nicotine) exposure can cause fetal IUGR and overexposure to maternal glucocorticoid (GC) in the uterus.As we known, insulin resistance is the common physiopathologic basis of metabolic syndrome. In adult, GC is the most common hormone which leads to insulin resistance. The mechanism is closely associated with the tissue specific expression of 11β-hydroxysteroid dehydrogenase (11β-HSD) and the glucocorticoid receptor (GR) in peripheral tissues, and it is also related to the alteration of the metabolic-related signal pathway. In the fetal period, the main metabolic-related pathways responsible for insulin resistance include:insulin and insulin-like growth factor (IGF) signal pathway (IGF-1, IGF-2, IR, IRS, AKT, and GLUT4), adiponectin signal pathway (adiponectin, AdiporR, AMPK) and leptin signal pathway (leptin, OB-R, JAK-2, AMPK). Little data is available to demonstrate whether these signal pathways are changed in the peripheral tissues of IUGR fetus. Metabonomics research based on nuclear magnetic resonance (NMR) is capable to detect the slight difference of various micromolecule metabolites in both normal and pathological states and find the different metabolites related the diseases through NMR spectras of body fluid, cellules and tissues. It has been widely used in systemic biology and translational medicine right now. However, the application of metabonomics in developmental toxicology is rare.The changes of metabolic-related signal pathways in the peripheral tissues of IUGR fetus caused by prenatal nicotine exposure as well as metabolins in body fluid are still not clarified. Therefore, in the present study, with the aim to confirm the mechanism of metabolic programming alternation in IUGR fetus induced by prenatal nicotine exposure, and provide the evidence to explain the fetal developmental origins of metabolic syndrome, we will investigate the specific expression of 11β-HSD/GR and the changes of metabolic-related signal pathways in the peripheral tissues (liver and skeletal muscle) of IUGR fetus induced by prenatal nicotine exposure via the methods of molecular biology. Moreover, we also use the metabonomics research methods based on NMR to observe the metabolome changes of biological fluids (fetal blood and amniotic fluid derived from IUGR fetus, maternal blood). The research will make a fully understanding in the risk factors of metabolic syndrome and provide guidance for good prenatal and postnatal care, and also give the new thoughts and new techniques to further exploration of the biomarkers of early clinical diagnosis for IUGR.Part One:Effect of prenatal nicotine exposure on 11β-HSD/GR mRNA expression and the metabolic-related signal pathways in the peripheral tissues of IUGR fetusObjectives:To investigate effect of prenatal nicotine exposure on 11β-HSD/GR mRNA expression and the metabolic-related signal pathways in the peripheral tissues (liver and skeletal muscle) of IUGR fetus via the methods of molecular biology. Methods:Pregnant rats were treated with 0.5,1.0 and 2.0 mg/kg nicotine respectively from GD 12. The control group was sham-treated with saline. On GD 20, the pregnant rats were sacrificed by decapitation with isoflurane anesthesia. Fetal body weights were recorded and fetal plasma specimens were collected. Fetal livers and skeletal muscles specimens were excised. The enzyme linked immunosorbent assay (ELISA) kits were used to detect the levels of fetal plasma adiponectin and corticosterone. Real-time quantitative PCR was used to detect mRNA expressions of the 11β-HSD-1/2 and GR; the signal pathway related genes of IGF insulin and insulin pathway (IGF-1, IGF-1R, IR, IRS-1/2, AKT, and GLUT4), adiponectin signal pathway (AdipoR, AMPK) and leptin receptor (OB-Ra, OB-Rb). Results:①The model of IUGR:Comparing with the control group, the fetal body weights were decreased by 5.41%,9.31% and 17.41% (P<0.01) and the rate of IUGR were increased by 13.91%,25.22% and 73.91% respectively in nicotine-treated groups (P<0.01). The levels of fetal plasma corticosterone in middle and high dose groups were increased by 30% and 50% respectively.②The metabolism of GC in liver:The expression of GR and 11β-HSD-1 were increased obviously (P<0.05), but 11β-HSD-2 was in a decreased tendency. 11β-HSD-1/11β-HSD-2 expression ratio was obviously increased (P<0.01).③IGF-like insulin and insulin pathways:The IGF-1 and IGF-1R mRNA expressions were decreased in a dose-dependent manner (P＜0.05, P＜0.01); IR was decreased in the high dose group (P<0.05).④Liver adiponectin and leptin pathways: Fetal plasma adiponectin in the middle and high dose groups were decreased by 16% and 15.7% respectively (P<0.05), the AdipoR had an increase tendency, AMPK increased in dose-dependent manner (P＜0.01), OB-Ra mRNA expression in middle and high dose groups were increased by 2.1 and 1.8 folds respectively (P<0.01), OB-Rb mRNA expression in high dose group was increased obviously (P＜0.05).⑤The metabolism of GC in skeleton muscle: 11β-HSD-1 mRNA expressions was in an ascendant tendency, and 11β-HSD-2 in high dose group was decreased obviously (P<0.05). The expression rate of 11β-HSD-1/11β-HSD-2 was obviously increased (P<0.01). GR expression was in an increased trend.⑥IGF-like insulin and insulin pathway in skeleton muscle:IGF-1R mRNA expression in the fetal skeleton muscle was decreased by 37%,42% and 41.5% respectively in a dose dependant manner (P＜0.05, P＜0.01), and the IRS-1 was also decreased obviously (P<0.05). Conclusion:High GC concentration in IUGR fetal circulation can enhance the activity of GC in liver and muscle via increasing 11β-HSD-1/11β-HSD-2 expression ratio as well as GR expression. The local high GC activity can inhibit the IGF/insulin signal pathway, and contribute to the development of adiponectin and leptin pathways. These lead to the alternation of metabolic programming of glucose and lipid, and these alternations will slow down the growth and development of fetus, eventually lead to IUGR. This mechanism is probably responsible for insulin resistance and increased susceptibility of metabolic syndrome in later adulthood.Part Two:Effect of prenatal nicotine exposure on metabonomics in fetal blood, amniotic fluid and maternal bloodObjectives:To investigate the effect of prenatal nicotine exposure on metabonomics in fetal blood, amniotic fluid and maternal blood using the metabonomics techniques based on NMR, and clarify the alternation of metabolic programming in fetal peripheral tissues induced by nicotine, and obtain a group of specific micromolecule metabolites (candidate biomarkers) for diagnosing IUGR. Methods:Pregnant rats were treated with 0.5,1.0 and 2.0 mg/kg nicotine respectively from GD 12. The control group was sham-treated with saline. On GD 20, the pregnant rats were sacrificed by decapitation after isoflurane anesthesia. Fetal plasma specimens were collected. Amniotic fluid and maternal plasma were collected. After pre-treatment, the samples were deal with NMR test. We used PCA and OPLS-DA methods to analysis the NMR data. Results:Compared with the control group, the 1H NMR spectrum of the fetal blood, amniotic fluid and maternal blood showed significant variation in metabolites of glucose, lipid and amino acid metabolism in each nicotine group. The result of PCA and OPLS-DA showed that,①in fetal plasma:In the low and middle dose groups, the increased substances included:lactic acid, Glycerol phosphatidylcholine, N-acetyl glycoprotein. The decreased substances include:VLDL, LDL, triglyceride, choline phosphate, leucine, isoleucine and tryptophan. In high dose group, the increased substances included:lactic acid, LDL, choline phosphate, and the decreased substances included:glucose, VLDL, glycerol phosphatidylcholine, leucine, isoleucine, glutamine and N-acetyl glycoprotein.②in amniotic fluid:The contents of lactic acid and pyruvic acid increased, while valine, leucine, isoleucine, threonine, methionine, lysine and tryptophan decreased.③in maternal plasma:In low and middle dose groups, the increased substances included:lactic acid, VLDL, HDL, glycerol phosphatidylcholine, phenylalanine, histidine, N-acetyl glycoprotein, and the decreased substances included:LDL, triglyceride, leucine. In addition, in low dose group, tyrosine, glutaminate and glutamine increased, but acetone decreased. In middle group, tryptophane and isoleucine decreased. In high dose group, the increased substances include:triglyceride and acetoacetic acid, and the decreased substances include:lactic acid, VLDL, LDL, glycerol phosphatidylcholine, leucine, isoleucine, lysine, glutamine, glutaminate, N-acetyl glycoprotein. Conclusion:Prenatal nicotine exposure can lead to the disorder of glycometabolism, lipid metabolism and amino acid metabolism of IUGR fetus. It mainly manifested in the following aspects:①glycometabolism:the anaerobic glycolysis of glucose increased and the aerobic metabolism decreased;②lipid metabolism:the synthesis of some lipoproteins such as VLDL and LDL decreased and the metabolism of glycerol phosphatidylcholine was disturbed;③amino acid metabolism:the shortages of some essential amino acid such as leucine, isoleucine and tryptophan, the use of glutamic acid suppressed, and the catabolism of protein increased. Lactic acid, VLDL, LDL, glycerol phosphatidylcholine, leucine, isoleucine, tryptophan, tyrosine and N-acetyl glycoprotein in maternal blood probably are the candidate biomarkers for diagnosing IUGR. |
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