| Metabolic syndrome is a collection of multiple diseases, including hypertension, hyperglycemia, blood fat disorder and obesity, which directly lead to fatty liver, diabetes, cardiovascular and cerebrovascular diseases. In china, the prevalence of people older than 20 years old has reached to 14%-16%. Insulin resistance means the target organs of insulin action become insensitive to insulin itself. It is not just a central event in many related metabolic disorders, but the common pathophysiological basis of metabolic syndrome. Although the identified symptom would not manifest until adulthood as well as middle or old age, the origin of disease usually could be traced back to childhood and infantile stage, even the fetal stage. Intrauterine growth retardation (IUGR) refers to either the fetus, with gestational age exceeding 37 weeks, weighs less than 2500 grams or the birth weight lies below 10th percentile for that gestational age, and the Morbidity of IUGR in China is about 7.5%. Epidemiological investigation shows that, between IUGR fetus and normal individual, the incidence of metabolic syndrome when grown up is 2.53 versus 1. Moreover, small for gestational age (SGA) babies caused by IUGR have 7 to 10 time higher incidence of metabolic syndrome than those who are appropriate for gestational age. All of the above suggest that there is a fetal origin of metabolic syndrome. Meanwhile, IUGR babies, a high risk group susceptible to metabolic syndrome, has drawn the most widespread attentions within medicine.Growing studies show that, for adult, high plasma GC concentration is one of the considerable regulatory factors of insulin resistance and metabolic syndrome. Adrenal gland is known as an important organ where GC can be synthesized and excreted. The action of GC to the target tissues not only depends on GC concentration in circulation, but also correlates with 11 P-hydroxysteroid dehydrogenase (11β-HSD) and glucocorticoid receptor (GR) in tissues. Both of them are characterized by mediating GC metabolism.11β-HSD-1 and 11β-HSD-2 could facilitate the biotransformation between inactive 17-hydroxy-11-dehydrocorticosterone and active cortisol. It is demonstrated that the distribution of 11β-HSD-1 and 11β-HSD-2 has tissue specificity.Placental 11β-HSD-2 could inactivate GC thereby protect the fetus from maternal GC while 11β-HSD-1 activate GC to accelerate fetal maturation in the third trimester of pregnancy and initiate delivery. Previous studies suggested that, a variety of adverse intrauterine environments (e.g. maternal stress) could decrease placental 11β-HSD-2 expression and thus open the placental barrier. Finally, the fetus was overexposed to maternal GC. Hypothalamic-pituitary-adrenal (HPA) axis plays an important role in stress response and hippocampus is one of the key components of HPA functional regulatory center. The change of expressions of fetal hippocampal 11β-HSD-1 and 11β-HSD-2 induced variation of local GC metabolic activation, which probably indirectly influences the normal development of fetal HPA via affecting hippocampal function. It has been demonstrated that chronic exposure of fetus to high concentration GC selectively injured hippocampus and lead to degeneration and necrosis of neuron. However, the relation between fetal GC metabolic activation and peripheral metabolic programming has not been reported yet. Other investigations show that there are cross-linked regulations between high GC exposure to adult and peripheral insulin resistance related signal pathways. The change of expressions of peripheral 11β-HSD-1 and 11β-HSD-2 could affect local GC metabolic activation and enhance GR function, which lead to insulin resistance in peripheral tissues and metabolic syndrome.Caffeine is a xanthine alkaloid widely present in coffee, tea, cola beverage and some analgesic drugs. Both clinical investigation and animal test demonstrate that, caffeine ingestion during pregnancy could lead to reproductive and embryo toxicity, besides, when ingested by child and adolescent, individual obesity incidence and adult metabolic syndrome susceptibility are significantly increased. All the proofs implicate that caffeine ingestion during pregnancy could induce embryo developmental toxicity and increase the susceptibility of adult metabolic syndrome. It is also considered as one of the most definite and dangerous inducement of IUGR. However, a series of scientific questions are proposed. Could caffeine exposure during pregnancy bring about the adult insulin resistance and metabolic disorders of IUGR fetus? And is there any intrauterine origin of it? Would caffeine exposure during pregnancy change the placental 11β-HSD-1/11β-HSD-2 expression and cause high maternal GC exposure to fetus? Furthermore, could high maternal GC exposure to fetus alter the functional development of fetal HPA axis and metabolic programming in peripheral tissues? Is there any epigenetic modification present in the caffeine mediated regulation of 11β-HSD-2 expression? Basing on the above, this project aims to establish IUGR as well as consequent adult insulin resistance rat model and to systematically study a series of parameters of IUGR fetus, including GC concentration in fetal blood, the function of HPA axis and its regulatory center hippocampus. Meanwhile, from the perspective of 11β-HSD-2 methylation, we plan to investigate caffeine induced fetal hippocampal GC metabolism and epigenetic mechanisms of GR functional change. The significance of this research is that, it illuminates the mechanism of functional development of fetal HPA axis and interprets the fetal origin of metabolic syndrome. All of these contribute to completely understand the new risk factor of adult metabolic syndrome and provide guidance of prepotency.PART ONEInsulin Resistance and Metabolic Disorders in Adult Caffeine Induced IUGR RatsObjective:To demonstrate caffeine exposure during pregnancy could lead to IUGR and consequent insulin resistance and metabolic disorders after maturation on rat model. Methods: The animals were divided into two main groups. The rats in group 1 were used to establish IUGR model induced by caffeine exposure during trimester of gestation (20-180 mg/kg-d). On day 20 of gestation (GD 20), the females were anesthetized with isoflurane and the fetuses were removed from uteri by cesarean section, dried of amniotic fluid, weighed and examine the length of body as well as tail. The incidence of IUGR was calculated later. All the needful organs or tissues were isolated and stored at-80℃immediately. The rats in group 2 were used to establish IUGR model by caffeine exposure (120 mg/kg-d), the dams were maintained for normal delivery. The weight gains of fetuses were recorded each day after birth. Blood specimen was collected on postnatal day 100 (PD 100), then, the serum was prepared to detect basic concentration of total cholesterol (TCH), triglyceride (TG), free fatty acid (FFA), glucose and insulin in circulation. Consequently, the oral glucose tolerance test (OGTT) was performed to calculate insulin resistance index. Results:For group 1, different dose of caffeine (20-180 mg/kg-d) significantly reduce the fetal weight, body length and tail length (P<0.01), the incidence of IUGR was increased as well (P<0.01), all results presented favorable dose-effect relationship. For group 2, the fetuses from caffeine treated dams had lower weight gain than those from normal control dams (P<0.05; P<0.01), and there is no changes in weight gain rate compared with normal control. The concentrations of TCH, TG and FFA in blood, without any gender specificity in adult offspring, were augmented at different degree. However, blood insulin concentration was increased in adult female offspring but decreased in adult male offspring. The fundamental blood glucose concentration had no obvious change. OGTT result showed that the IRI of adult female offspring from caffeine treated dams was significantly increased comparing with those from normal dams (P<0.05), conversely, the IRI of adult male offspring was decreased (P<0.05). Conclusion: IUGR could be induced by caffeine exposure during trimester of gestation (20-180 mg/kg-d), suggesting the IUGR rat model was successfully established. Caffeine induced (120 mg/kg-d) IUGR fetuses didn't present "catch-up" obesity after birth but manifested metabolic disorders. Insulin resistance only existed in adult female offspring.PART TWOPrenatal caffeine exposure induced maternal GC overexposure to fetus and abnormality of fetal HPA axis developmentObjective:To explore the internal relations underlying a series of events, including prenatal caffeine exposure, high maternal GC concentration in fetal blood, functional change of HPA axis and its regulatory center hippocampus during fetal stage and different postnatal periods, and the sensibility of HPA axis to the stress in adult offspring. Discuss the potential mechanisms and biological significance of caffeine induced functional change of HPA axis.Methods:For the prenatal fetuses from group 1 dams, we used real-time quantitative PCR (RT-PCR) and western blotting technique to analysis the mRNA and protein expressions of parameters indicating the function of HPA axis, including hippocampus GR, 11β-HSD-1 and 11β-HSD-2; hypothalamic corticotropin releasing hormone (CRH); adrenal steroidogenic acute regulatory protein (StAR) and cytochrome P450 cholesterol side chain cleavage (P450scc); placental 11β-HSD-1 and 11β-HSD-2. The plasma concentrations of maternal corticosterone, and fetal ACTH and corticosterone were measured by ELISA kit. For postnatal offspring from group 2 dams, the plasma corticoserone and ACTH concentration, and the mRNA expressions of hippocampus GR were all measured at different time points, such as PD1, PD7, PD35 and PD60, using ELISA kit and RT-PCR, respectively. The adult offspring rats were allocated according to gender after postnatal day of 125 and all of them received mild chronic stimulus by means of ice water swimming test for 2 weeks, then calculated the increasing rate of plasma corticoserone and ACTH concentration, and the mRNA expression of hippocampus GR after ice water swimming. Results:For group 1, fetal hippocampal GR expression was significantly increased after caffeine (20-180 mg/kg-d) treatment (P<0.05). The expressions of hypothalamic CRH, fetal adrenal StAR and P450scc were all obviously decreased (P<0.05, P<0.01), but plasma concentrations of maternal corticoserone and fetal ACTH and corticoserone were all increased (P<0.05, P<0.01). Moreover,11β-HSD-1 mRNA expression was increased while 11β-HSD-2 mRNA expression was decreased (P<0.05). This phenomenon could be observed in both placenta and fetal hippocampus. For group 2, on PD 1 and PD 7, the plasma corticoserone and ACTH concentration in offspring rats from caffeine treated (120 mg/kg-d) dams was much higher than those from control dams. However, these high concentrations declined on PD 35 and dropped to a relatively low level on PD 100. Ice water swimming test showed that, the mRNA expression of hippocampus GR in female offspring in caffeine group, but not the male, is much more higher than the control after ice water swimming (P<0.05). Furthermore, the increasing rates of plasma corticoserone and ACTH, both in male and female offsprings in caffeine group, were higher than control (P<0.05). Conclusions:Prenatal caffeine exposure could cause maternal stress and/or increase the placental 11β-HSD-1/11β-HSD-2 expression ratio, impair the placental barrier to maternal GC and finally cause the over exposure of maternal GC to fetus. This terminal result would not only directly inhibit HPA activity by negative feedback regulation, but also increase the fetal hippocampal GR expression to achieve the same inhibition of HPA axis by negative feedback regulation. Therefore, the capability of steroid hormone synthesis of fetus itself is decreased and the functional development of HPA axis becomes slower. More than that, the effects of prenatal caffeine exposure on HPA axis function could be continued to postnatal stage even adulthood, suggesting it is one of the most important reasons for HPA axis has decreased basic level but increased sensibility to stress in adulthood.PART THREEPrenatal caffeine exposure induced the changes of metabolic pathways and metabolites in peripheral tissues of fetal ratsObjective:To systemically explore internal relations underlying a series of events, including prenatal caffeine exposure, high GC concentration in fetal blood, and change of insulin resistance related signal pathway in fetal peripheral tissue as well as metabolites of glucose, lipid and amino acid in blood. Demonstrate the variation of fetal peripheral metabolic programming and interpret the fetal origin of adult insulin resistance. Methods:Using real-time quantitative PCR to detect the variation of some key factors'expressions in each pathway, such as 11β-HSD-1,11β-HSD-2 as well as GR in hepatic and muscle GC metabolic pathway; IGF-1, IGF-1R, IR and IRS-1/2 in insulin signal pathway; adipoR2, OBRa, OBRb and AMPKa2 in adiponectin and leptin signal pathway. The adiponectin level of fetal blood was measured by ELISA kit. NMR technique was used to detect the variation of multiple metabolites in fetal blood. Results:The expressions of IGF-1, IGF-1R and IR in fetal liver and IR in fetal muscle were significantly decreased (P<0.05, P<0.01). In fetal liver, the expressions of AdipoR2, AMPKa2, OBRa and OBRb were all increased (P<0.05, P<0.01). However, the expressions of IRS-2 and IRS-1 in fetal liver and muscle had no significant change. The result of ELISA showed that the adiponectin level in fetal blood was obviously increased (P<0.05). The NMR analysis showed that the contents of multiple endogenous metabolites, such as glucose, amino acid and lipid metabolite, were altered. The main consequences include the contents of a series of products were significantly increased (P<0.05), including a-glucose, P-glucose, cholesterol, tyrosine, tryptophane and phenylalanine. Meanwhile, the contents of VLDL, TG, isoleucine and valine were obviously decreased (P<0.05). And the expressions of 11β-HSD-1 and GR in fetal liver and muscle were increased (P<0.05) while 11β-HSD-2 expression was decreased. Conclusions:High maternal GC concentration induced by prenatal caffeine exposure might active the GC metabolic activation and GR expression in peripheral tissues (liver and muscle), which in one hand inhibit the functional development of IGF parainsulin/insulin signal pathway and in the other hand enhance adiponectin signal pathway and leptin signal pathway so that the fetal metabolic programming is changed. These changes will retard the fetal growth and development directly and cause IUGR. This kind of fetal metabolic programming change will be continued to postnatal stage even adulthood, which could be recognized as the intrauterine basis of adult insulin resistance and metabolic disorders.PART FOUR11β-HSD-2 methylation mediated caffeine induced IUGR and adult insulin resistanceObjective:To explore internal relations underlying a series of events, including caffeine intervention, increased methylation of 11β-HSD-2 promoter,11β-HSD-1/11β-HSD-2 expression ratio was augmented, enhanced GC metabolic activation and intensified GR expression, on cultured fetal hippocampal neuron cell. Methods:Establishing primary fetal hippocampal neuron culture system in vitro. The cultured fetal hippocampal neurons were treated with 300μM caffeine for 0,6,12,24,48 and 72 h or with 0,0.3,3,30 and 300μM caffeine for 24 h. The mRNA and protein expressions of hippocampus GR,11β-HSD-1 and 11β-HSD-2 were determined by real-time quantitative RT-PCR and/or western blotting. DNA methylation microarray was utilized to analysis genome-wide profiling of promoter methylation pattern on cultured fetal hippocampal neuron cell after treated with 300μM caffeine for 24 h. Using bisulfite genomic sequencing PCR (BSP) to detect and analysis the methylation pattern of 11β-HSD-2 promoter. Results:Treatment with various dose of caffeine could significantly decrease mRNA expression of 11β-HSD-2 (P<0.05,P<0.01) while increase mRNA expression of 11β-HSD-1 as well as mRNA and protein expression of GR (P<0.05) in favorable time-correlated and dose-dependent manner. DNA methylation microarray results showed that, in caffeine treated hippocampal neuron cell, there was hypermethylation within -358--77 bp region of 11β-HSD-2 promoter. Further BSP results showed that, comparing with control, the total methylation rate of -358-77 bp region of 11β-HSD-2 promoter was significantly increased (P<0.05), the incidence of methylation on CpG site was obviously increased (P<0.05, P<0.01), especially at -220,-214,-211,-193,-167,-152,-130 and-111 bp. Conclusions:Caffeine could depress fetal hippocampal 11β-HSD-2 gene expression via increasing the total methylation frequency of its promoter region, which would inhibit inactivation of local GC and enhance 11β-HSD-1 expression as well as GR. The effects of caffeine on the methylation pattern of fetal hippocampal 11β-HSD-2 promoter might simultaneously exist in placenta and other fetal tissues such as liver and muscle, which probably an important reason to explain the enhanced GC metabolic activation and GR function in these tissues. In addition, it also might be the cause of IUGR as well as the intrauterine origin of adult insulin resistance.CONCLUSIONSCaffeine expoure during pregancy decreased the expressions of 11β-HSD-2 in placental and other fetal tissues, such as fetal hippocampus, liver, and muscle, through the increased total promoter region methylation frequency of 11β-HSD-2, and increased the 11β-HSD-1/11β-HSD-2 expression ratio. These would finally caused fetal over expoure to maternal GC via the impaired placental barrier to maternal GC, and promoted fetal HPA axis developmental anomaly and metabolic programming changes.Interestingly, these changes, essentially induced by caffeine exposure during pregnancy, would be continued to postnatal stage and even adulthood. The related manifestations in those stages are more likely to be the decreased basic function of HPA axis and the increased sensibility to stress, and metabolic disorders.
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