孕期咖啡因暴露所致子代成年代谢紊乱的胎儿发生机制
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摘要
代谢综合征是高血压、高血糖、血脂紊乱和肥胖等多种疾病在人体内集结的一种状态,可直接引起脂肪肝、糖尿病和心脑血管疾病,在我国城市20岁以上人群中的患病率已达14%-16%。胰岛素抵抗是其发生的共同病理生理基础。虽然胰岛素抵抗在成年或中老年才呈现明确的疾病状态,但其起病的根源往往可追溯至儿童、婴幼儿甚至胎儿时期。宫内生长迟缓(IUGR)是指孕周大于37周胎儿出生体重小于2500 g,或胎儿体重低于其孕龄平均体重的两个标准差。IUGR在我国发病率约7.5%。流行病学调查已表明,IUGR胎仔出现成年代谢综合征发生率是正常的2.53倍,IUGR引起的小于胎龄儿(SGA)的成年代谢综合征发生率较适于胎龄儿高出7-10倍,提示代谢综合征存在着一个胎儿起源。IUGR这个成年代谢综合征的高危群体已引起了医学界的广泛关注。
越来越多的研究表明,成人高血糖皮质激素(GC)水平是胰岛素抵抗及代谢综合征发生的重要调节因素。已知肾上腺是GC合成与分泌的重要器官,体内GC作用于靶组织不仅有赖于循环中GC浓度,而且与组织中介导GC代谢的11β-羟类固醇脱氢酶(11β-HSD)和糖皮质激素受体(GR)表达有关。11β-HSD-1和11β-HSD-2使无活性的17-羟-11-脱氢皮质酮和活性的皮质醇相互转化。研究表明,11β-HSD-1和11β-HSD-2在机体内分布具有组织特异性。
胎盘11β-HSD-2通过灭活GC可保护胎儿免受母源性GC干扰,而胎盘11p-HSD-1则通过活化GC诱导妊娠晚期胎儿成熟和分娩发动。研究提示,多种不良宫内环境(如孕妇应激)可降低胎盘11p-HSD-2的表达而开放胎盘屏障,使胎儿过暴露于母源性GC。下丘脑-垂体-肾上腺(HPA)轴在应激防御反应中发挥着重要作用。海马是HPA轴功能调节中枢的重要组成部分。胎海马11β-HSD-1和11β-HSD-2表达改变所致局部GC代谢活化变化,将可能通过影响海马功能而间接影响胎儿HPA的正常发育。研究已发现,胎儿长期暴露于高浓度GC可选择性损伤海马,导致神经元变性坏死。有关胎儿时期GC活化代谢与外周组织代谢编程之间的关系尚未见明确报道。研究发现,成人高GC暴露与外周组织胰岛素抵抗相关的代谢信号通路之间存在着网络调控。外周组织中11β-HSD-1和11β-HSD-2的表达改变,可通过影响局部GC代谢活化和GR功能增强,诱导外周组织胰岛素抵抗和代谢综合征的发生。
咖啡因(caffeine)属黄嘌呤类生物碱,广泛存在于咖啡、茶、可乐及一些镇痛药物中。临床及动物实验表明,孕期咖啡因摄入能引起生殖和胚胎毒性；儿童和青少年时期咖啡因摄入可显著增加个体肥胖和成年代谢综合征的易感性。提示,咖啡因孕期摄入可引起胚胎发育毒性及成年代谢综合征易感性增加,是IUGR最确切和危险的诱因之一。然而,孕期咖啡因暴露是否可引起IUGR胎儿成年后胰岛素抵抗和代谢综合征?IUGR胎儿成年后胰岛素抵抗和代谢综合征是否存在宫内起源?孕期咖啡因暴露是否会引起胎盘11β-HSD-1/11β-HSD-2表达改变和胎儿母源性高GC暴露?母源性高GC暴露是否会进一步引起胎儿HPA轴功能发育和外周组织代谢编程的改变?咖啡因对11p-HSD-2的表达调节是否存在表观遗传修饰现象?
为此,本课题将建立孕期咖啡因暴露所致大鼠IUGR及其成年胰岛素抵抗模型,系统研究IUGR胎儿血GC水平、HPA轴及其调节中枢海马功能、外周组织中胰岛素抵抗相关代谢信号通路关键蛋白表达及其体内代谢产物的变化；进一步从11β-HSD-2甲基化修饰角度,探讨咖啡因所致胎海马GC代谢及其GR功能变化的表观遗传机制,以阐明咖啡因引起胎儿HPA轴功能发育的发生机制,解析代谢综合征的胎儿发育起源,充分认识成年代谢综合征新的危险因素,指导优生优育。
第一部分孕期咖啡因暴露所致IUGR胎鼠成年胰岛素抵抗和代谢紊乱
目的：通过整体动物实验,证实孕期咖啡因暴露可致胎鼠IUGR和成年鼠胰岛素抵抗和代谢紊乱发生。方法：第一批动物：建立孕中晚期咖啡因(20-180 mg/kg-d)暴露所致大鼠IUGR模型。在临产前一天(GD20)异氟醚麻醉状态下取胎鼠,检测胎鼠体重、身长和尾长,计算IUGR发生率。第二批动物：建立孕中晚期咖啡因(120 mg/kg-d)暴露所致大鼠IUGR模型后让其自然生产,记录仔鼠出生后体重增长情况,出生后100天(PD100)取血制备血清,检测血总胆固醇(TCH)、甘油三酯(TG)、游离脂肪酸(FFA)、血糖和血胰岛素的基础浓度变化,进一步进行口服葡萄糖耐量实验(OGTT)并计算胰岛素抵抗指数(IRI)。结果：第一批动物：不同剂量咖啡因(20-180 mg/kg-d)可明显降低胎鼠的体重、身长和尾长(P<0.01),增加胎鼠的IUGR发生率(P<0.01),表现为良好的量效关系。第二批动物：①咖啡因处理组仔鼠出生后体重增长显著低于正常对照组(P<0.05,P<0.01),体重增长率则与正常对照组无显著差异。②在咖啡因处理组的代谢综合征相关基础指标中,血TCH、TG和FFA浓度无论在雄性成年鼠还是雌性成年鼠,皆出现不同程度的升高；血胰岛素浓度在雌性成年鼠增加,但在雄性成年鼠降低；基础血糖均无明显变化。③OGTT结果表明,与正常对照组相比,咖啡因处理组雌性成年鼠IRI增加(P<0.05),然而雄性成年鼠IRI却表现为降低(P<0.05)。结论：孕中晚期咖啡因(20-180 mg/kg·d)暴露可致大鼠IUGR发生,说明IUGR造模成功。咖啡因(120 mg/kg·d)所致的IUGR仔鼠出生后未出现追赶性肥胖,成年后仍表现代谢紊乱,其中雌性成年鼠出现胰岛素抵抗。
第二部分孕期咖啡因暴露所致胎鼠母源性GC过暴露和HPA轴发育异常
目的：在咖啡因所致的IUGR胎鼠(第一批动物)及其成年鼠胰岛素抵抗(第二批动物)模型上,研究孕期咖啡因暴露-母源性胎血高GC-胎鼠HPA轴及其调节中枢海马功能改变-出生后不同时间HPA轴及其调节中枢海马功能改变-成年鼠HPA轴应激敏感性变化之间的内在联系,探讨咖啡因所致HPA轴功能改变的发生机制及其生物学意义。方法：采用实时定量RT-PCR和Western blot技术,检测出生前胎鼠(GD20)海马(11β-HSD-1和11β-HSD-2)、下丘脑(促肾上腺皮质激素释放激素CRH)、肾上腺(甾体合成急性调节蛋白StAR和P450胆固醇侧链裂解酶P450scc)、胎盘(11β-HSD-1、11β-HSD-2)上与胎儿HPA轴功能相关指标的mRNA和蛋白表达；ELISA试剂盒检测母鼠、胎鼠(GD20)和出生后仔鼠不同生长时间点(PD1、PD7、PD35和PD60)血皮质酮和ACTH浓度；实时定量RT-PCR检测胎鼠(GD20)和出生后仔鼠不同生长时间点(PD1、PD7、PD35和PD60)GR的mRNA表达；PD125的成年鼠分雌、雄进行持续2周的冰水游泳刺激实验,采用ELISA试剂盒及实时定量RT-PCR技术,分别检测刺激前、后血皮质酮和ACTH浓度以及刺激后海马GR的mRNA表达。结果：①第一批动物：在咖啡因(20-180 mg/kg·d)处理组,胎盘11β-HSD-1表达显著升高(P<0.05),11β-HSD-2表达显著降低(P<0.05)；母血皮质酮水平出现显著升高(P<0.01)；胎血皮质酮和ACTH浓度均出现显著性升高(P<0.05)；进一步发现,下丘脑CRH、胎肾上腺StAR和P450scc表达显著降低(P<0.05,P<0.01),胎海马11β-HSD-1和GR表达显著增加(P<0.05),1β-HSD-2表达出现显著性降低(P<0.05)。②第二批动物：在咖啡因(120 mg/kg·d)处理组,PD1和PD7的动物血皮质酮和ACTH浓度高于正常对照组,从PD35开始呈现降低趋势,至PD60达到较低水平；海马GR表达持续显著高于正常对照组(P<0.05)；冰水游泳刺激实验发现,雌性仔鼠海马GR表达在刺激后较正常对照组显著降低(P<0.05),雄性仔鼠则无明显改变。进一步发现,冰水游泳刺激前后雌、雄成年仔鼠血浆皮质酮和ACTH浓度增长率较正常对照组均出现显著性升高,以雌性最为明显。结论：孕期咖啡因暴露能通过母体应激和/或增加胎盘11β-HSD-1/11β-HSD-2的表达比,削弱胎盘对母源性GC的屏障作用,引起胎儿“母源性GC过暴露”。后者通过诱导胎海马GR表达,负反馈抑制胎HPA轴的活性,从而使胎儿自身甾体激素合成能力进一步降低,HPA轴功能发育减慢。孕期咖啡因对胎鼠海马GR和HPA轴功能的影响可一直延续至出生后甚至成年,是其成年HPA轴基础水平降低但应激反应增强的重要原因。
第三部分孕期咖啡因暴露所致胎鼠外周组织代谢通路及代谢产物改变
目的：在咖啡因所致的IUGR胎鼠上,系统研究孕期咖啡因暴露-胎血高GC-外周组织GC活化代谢-外周组织胰岛素抵抗相关代谢信号通路改变-胎血糖、氨基酸和脂肪代谢产物变化”之间的内在联系,证实胎鼠外周组织代谢编程改变,解析成年胰岛素抵抗的胎儿发生机制。方法：采用实时定量RT-PCR技术,检测肝脏和骨骼肌GC活化代谢途径(11β-HSD-1、11β-HSD-2和GR)、胰岛素样生长因子和胰岛素信号通路(胰岛素样生长因子IGF-1、胰岛素样生长因子-1受体IGF-1R、胰岛素受体IR、胰岛素受体底物IRS-1/2、蛋白激酶B PKB/Akt2、葡萄糖转运体4 GLUT4)、脂联素和瘦素信号通路(脂联素受体2 AdipoR2、a型瘦素受体OBRa、b型瘦素受体OBRb、AMP激酶α2 AMPKa2)的表达变化。ELISA试剂盒检测胎血脂联素水平。基于核磁共振(NMR)的代谢组学技术用于检测胎血中糖代谢、脂代谢和氨基酸代谢产物的变化。结果：①胰岛素抵抗相关代谢信号通路：咖啡因处理组可显著降低胎肝IGF-1、IGF-1R、IR的表达以及胎骨骼肌IR的表达(P<0.05,P<0.01),升高胎肝AdipoR2、OBRa、OBRb和AMPKa2的表达(P<0.05,P<0.01),但胎肝和骨骼肌中IRS-2、IRS-1、Akt2和GLUT4的表达变化不明显。ELISA结果显示,咖啡因处理组可显著升高胎血脂联素水平(P<0.05)；②NMR检测和分析结果显示,咖啡因处理组可引起IUGR胎儿内生的多种糖、氨基酸和脂肪代谢产物含量改变,主要表现为：胎血中α-葡萄糖、β-葡萄糖、胆固醇、酪氨酸、色氨酸和苯丙氨酸含量显著性升高(P<0.05),极低密度脂蛋白、TG、异亮氨酸和缬氨酸含量显著性降低(P<0.05)；③GC活化代谢：咖啡因处理组的胎肝和骨骼肌均出现11β-HSD-1、GR表达升高(P<0.05),但11β-HSD-2表达降低。结论：孕期咖啡因所致“母源性高GC”可能通过激活外周组织(肝脏和骨骼肌)的GC活化代谢和GR表达,一方面抑制IGF类胰岛素/胰岛素信号通路的功能发育,另一方面促进脂联素信号通路和瘦素信号通路功能增强,从而使胎鼠代谢编程发生改变,NMR技术也证实孕期咖啡因暴露可导致胎鼠内生的多种糖、脂肪和氨基酸代谢产物含量改变。这些变化将直接减慢胎儿的生长发育速度,引起IUGR发生。咖啡因孕期暴露所致胎鼠代谢编程改变将一直延续至出生后甚至成年,成为成年胰岛素抵抗和代谢紊乱的宫内起端。
第四部分11β-HSD-2甲基化修饰改变介导咖啡因所致胎海马GC活化代谢和GR高表达
目的：在细胞水平,以胎海马神经元为代表,通过研究咖啡因处理-11β-HSD-2启动子甲基化增加-11β-HSD-1/11β-HSD-2表达比增加-GC活化代谢增强及GR表达增加之间的内在联系,证实11β-HSD-2甲基化修饰改变介导咖啡因所致胎海马GC活化代谢和GR高表达,探讨咖啡因引起IUGR及其成年代谢紊乱发生的表观遗传机制。方法：建立原代胎海马神经元体外培养系统,以0-300μM咖啡因处理胎海马神经元24 h,或以300μM咖啡因处理0、6、12、24、48和72 h,实时定量RT-PCR和/或Western blot技术检测海马神经元GR、11β-HSD-1和11p-HSD-2的mRNA和蛋白表达；商业化启动子甲基化芯片筛选300μM咖啡因处理胎海马神经元24 h后全基因启动子区的甲基化情况,亚硫酸氢盐修饰后测序(BSP)技术检测和分析11β-HSD-2启动子区的甲基化频率。结果：①GC活化代谢：不同浓度咖啡因处理能显著降低11β-HSD-2的mRNA表达(P<0.05,P<0.01),升高11β-HSD-1的mRNA表达以及GR的mRNA和蛋白表达(P<0.05)。皆呈现良好的时间相关性和浓度依赖性。②11p-HSD-2的甲基化修饰：甲基化芯片筛选发现,咖啡因处理组发生甲基化修饰的基因共1430个,发生去甲基化修饰基因共2337个。其中,海马11β-HSD-2启动子区-358--77 bp区域发生了显著的高甲基化发生率,为正常对照组的2.51倍。进一步的BSP验证结果显示,咖啡因组11β-HSD-2启动子区-358--77区域总甲基化率较对照组显著增加(P<0.05),在-220、-214、-211、-193、-167、-152、-130、-111bp处的CpG位点甲基化发生率均出现显著升高(P<0.05,P<0.01)。结论：咖啡因可能通过增加胎海马11p-HSD-2启动子区总甲基化频率,降低11β-HSD-2表达,由此抑制局部GC灭活代谢而诱导11β-HSD-1表达增加,后者将诱导GR表达增加。咖啡因对胎海马11β-HSD-2启动子区甲基化修饰的直接改变,还可能同时存在于胎盘和其他胎儿组织(如胎肝、胎骨骼肌),可能是引起这些组织GC活化代谢及GR功能增强的重要原因,也是最终引起IUGR及其成年胰岛素抵抗的宫内发生起端。
孕期咖啡因暴露通过增加胎盘和多种胎组织(如胎海马、肝脏和骨骼肌)11β-HSD-2启动子区的总甲基化频率并降低其表达,增加这些组织11β-HSD-1/11β-HSD-2的表达比,由此削弱胎盘对母源性GC的屏障作用,引起胎儿母源性GC过暴露,并促使胎HPA轴发育异常和外周组织代谢编程改变。孕期咖啡因暴露所致胎儿HPA轴功能和代谢编程改变还将延续至出生后甚至成年,表现为成年后基础HPA轴功能降低、应激反应敏感性增加以及代谢紊乱的发生。
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 ONE
Insulin Resistance and Metabolic Disorders in Adult Caffeine Induced IUGR Rats
Objective: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 TWO
Prenatal caffeine exposure induced maternal GC overexposure to fetus and abnormality of fetal HPA axis development
Objective: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 THREE
Prenatal caffeine exposure induced the changes of metabolic pathways and metabolites in peripheral tissues of fetal rats
Objective: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 FOUR
11β-HSD-2 methylation mediated caffeine induced IUGR and adult insulin resistance
Objective: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.
CONCLUSIONS
Caffeine 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.
越来越多的研究表明,成人高血糖皮质激素(GC)水平是胰岛素抵抗及代谢综合征发生的重要调节因素。已知肾上腺是GC合成与分泌的重要器官,体内GC作用于靶组织不仅有赖于循环中GC浓度,而且与组织中介导GC代谢的11β-羟类固醇脱氢酶(11β-HSD)和糖皮质激素受体(GR)表达有关。11β-HSD-1和11β-HSD-2使无活性的17-羟-11-脱氢皮质酮和活性的皮质醇相互转化。研究表明,11β-HSD-1和11β-HSD-2在机体内分布具有组织特异性。
胎盘11β-HSD-2通过灭活GC可保护胎儿免受母源性GC干扰,而胎盘11p-HSD-1则通过活化GC诱导妊娠晚期胎儿成熟和分娩发动。研究提示,多种不良宫内环境(如孕妇应激)可降低胎盘11p-HSD-2的表达而开放胎盘屏障,使胎儿过暴露于母源性GC。下丘脑-垂体-肾上腺(HPA)轴在应激防御反应中发挥着重要作用。海马是HPA轴功能调节中枢的重要组成部分。胎海马11β-HSD-1和11β-HSD-2表达改变所致局部GC代谢活化变化,将可能通过影响海马功能而间接影响胎儿HPA的正常发育。研究已发现,胎儿长期暴露于高浓度GC可选择性损伤海马,导致神经元变性坏死。有关胎儿时期GC活化代谢与外周组织代谢编程之间的关系尚未见明确报道。研究发现,成人高GC暴露与外周组织胰岛素抵抗相关的代谢信号通路之间存在着网络调控。外周组织中11β-HSD-1和11β-HSD-2的表达改变,可通过影响局部GC代谢活化和GR功能增强,诱导外周组织胰岛素抵抗和代谢综合征的发生。
咖啡因(caffeine)属黄嘌呤类生物碱,广泛存在于咖啡、茶、可乐及一些镇痛药物中。临床及动物实验表明,孕期咖啡因摄入能引起生殖和胚胎毒性；儿童和青少年时期咖啡因摄入可显著增加个体肥胖和成年代谢综合征的易感性。提示,咖啡因孕期摄入可引起胚胎发育毒性及成年代谢综合征易感性增加,是IUGR最确切和危险的诱因之一。然而,孕期咖啡因暴露是否可引起IUGR胎儿成年后胰岛素抵抗和代谢综合征?IUGR胎儿成年后胰岛素抵抗和代谢综合征是否存在宫内起源?孕期咖啡因暴露是否会引起胎盘11β-HSD-1/11β-HSD-2表达改变和胎儿母源性高GC暴露?母源性高GC暴露是否会进一步引起胎儿HPA轴功能发育和外周组织代谢编程的改变?咖啡因对11p-HSD-2的表达调节是否存在表观遗传修饰现象?
为此,本课题将建立孕期咖啡因暴露所致大鼠IUGR及其成年胰岛素抵抗模型,系统研究IUGR胎儿血GC水平、HPA轴及其调节中枢海马功能、外周组织中胰岛素抵抗相关代谢信号通路关键蛋白表达及其体内代谢产物的变化；进一步从11β-HSD-2甲基化修饰角度,探讨咖啡因所致胎海马GC代谢及其GR功能变化的表观遗传机制,以阐明咖啡因引起胎儿HPA轴功能发育的发生机制,解析代谢综合征的胎儿发育起源,充分认识成年代谢综合征新的危险因素,指导优生优育。
第一部分孕期咖啡因暴露所致IUGR胎鼠成年胰岛素抵抗和代谢紊乱
目的：通过整体动物实验,证实孕期咖啡因暴露可致胎鼠IUGR和成年鼠胰岛素抵抗和代谢紊乱发生。方法：第一批动物：建立孕中晚期咖啡因(20-180 mg/kg-d)暴露所致大鼠IUGR模型。在临产前一天(GD20)异氟醚麻醉状态下取胎鼠,检测胎鼠体重、身长和尾长,计算IUGR发生率。第二批动物：建立孕中晚期咖啡因(120 mg/kg-d)暴露所致大鼠IUGR模型后让其自然生产,记录仔鼠出生后体重增长情况,出生后100天(PD100)取血制备血清,检测血总胆固醇(TCH)、甘油三酯(TG)、游离脂肪酸(FFA)、血糖和血胰岛素的基础浓度变化,进一步进行口服葡萄糖耐量实验(OGTT)并计算胰岛素抵抗指数(IRI)。结果：第一批动物：不同剂量咖啡因(20-180 mg/kg-d)可明显降低胎鼠的体重、身长和尾长(P<0.01),增加胎鼠的IUGR发生率(P<0.01),表现为良好的量效关系。第二批动物：①咖啡因处理组仔鼠出生后体重增长显著低于正常对照组(P<0.05,P<0.01),体重增长率则与正常对照组无显著差异。②在咖啡因处理组的代谢综合征相关基础指标中,血TCH、TG和FFA浓度无论在雄性成年鼠还是雌性成年鼠,皆出现不同程度的升高；血胰岛素浓度在雌性成年鼠增加,但在雄性成年鼠降低；基础血糖均无明显变化。③OGTT结果表明,与正常对照组相比,咖啡因处理组雌性成年鼠IRI增加(P<0.05),然而雄性成年鼠IRI却表现为降低(P<0.05)。结论：孕中晚期咖啡因(20-180 mg/kg·d)暴露可致大鼠IUGR发生,说明IUGR造模成功。咖啡因(120 mg/kg·d)所致的IUGR仔鼠出生后未出现追赶性肥胖,成年后仍表现代谢紊乱,其中雌性成年鼠出现胰岛素抵抗。
第二部分孕期咖啡因暴露所致胎鼠母源性GC过暴露和HPA轴发育异常
目的：在咖啡因所致的IUGR胎鼠(第一批动物)及其成年鼠胰岛素抵抗(第二批动物)模型上,研究孕期咖啡因暴露-母源性胎血高GC-胎鼠HPA轴及其调节中枢海马功能改变-出生后不同时间HPA轴及其调节中枢海马功能改变-成年鼠HPA轴应激敏感性变化之间的内在联系,探讨咖啡因所致HPA轴功能改变的发生机制及其生物学意义。方法：采用实时定量RT-PCR和Western blot技术,检测出生前胎鼠(GD20)海马(11β-HSD-1和11β-HSD-2)、下丘脑(促肾上腺皮质激素释放激素CRH)、肾上腺(甾体合成急性调节蛋白StAR和P450胆固醇侧链裂解酶P450scc)、胎盘(11β-HSD-1、11β-HSD-2)上与胎儿HPA轴功能相关指标的mRNA和蛋白表达；ELISA试剂盒检测母鼠、胎鼠(GD20)和出生后仔鼠不同生长时间点(PD1、PD7、PD35和PD60)血皮质酮和ACTH浓度；实时定量RT-PCR检测胎鼠(GD20)和出生后仔鼠不同生长时间点(PD1、PD7、PD35和PD60)GR的mRNA表达；PD125的成年鼠分雌、雄进行持续2周的冰水游泳刺激实验,采用ELISA试剂盒及实时定量RT-PCR技术,分别检测刺激前、后血皮质酮和ACTH浓度以及刺激后海马GR的mRNA表达。结果：①第一批动物：在咖啡因(20-180 mg/kg·d)处理组,胎盘11β-HSD-1表达显著升高(P<0.05),11β-HSD-2表达显著降低(P<0.05)；母血皮质酮水平出现显著升高(P<0.01)；胎血皮质酮和ACTH浓度均出现显著性升高(P<0.05)；进一步发现,下丘脑CRH、胎肾上腺StAR和P450scc表达显著降低(P<0.05,P<0.01),胎海马11β-HSD-1和GR表达显著增加(P<0.05),1β-HSD-2表达出现显著性降低(P<0.05)。②第二批动物：在咖啡因(120 mg/kg·d)处理组,PD1和PD7的动物血皮质酮和ACTH浓度高于正常对照组,从PD35开始呈现降低趋势,至PD60达到较低水平；海马GR表达持续显著高于正常对照组(P<0.05)；冰水游泳刺激实验发现,雌性仔鼠海马GR表达在刺激后较正常对照组显著降低(P<0.05),雄性仔鼠则无明显改变。进一步发现,冰水游泳刺激前后雌、雄成年仔鼠血浆皮质酮和ACTH浓度增长率较正常对照组均出现显著性升高,以雌性最为明显。结论：孕期咖啡因暴露能通过母体应激和/或增加胎盘11β-HSD-1/11β-HSD-2的表达比,削弱胎盘对母源性GC的屏障作用,引起胎儿“母源性GC过暴露”。后者通过诱导胎海马GR表达,负反馈抑制胎HPA轴的活性,从而使胎儿自身甾体激素合成能力进一步降低,HPA轴功能发育减慢。孕期咖啡因对胎鼠海马GR和HPA轴功能的影响可一直延续至出生后甚至成年,是其成年HPA轴基础水平降低但应激反应增强的重要原因。
第三部分孕期咖啡因暴露所致胎鼠外周组织代谢通路及代谢产物改变
目的：在咖啡因所致的IUGR胎鼠上,系统研究孕期咖啡因暴露-胎血高GC-外周组织GC活化代谢-外周组织胰岛素抵抗相关代谢信号通路改变-胎血糖、氨基酸和脂肪代谢产物变化”之间的内在联系,证实胎鼠外周组织代谢编程改变,解析成年胰岛素抵抗的胎儿发生机制。方法：采用实时定量RT-PCR技术,检测肝脏和骨骼肌GC活化代谢途径(11β-HSD-1、11β-HSD-2和GR)、胰岛素样生长因子和胰岛素信号通路(胰岛素样生长因子IGF-1、胰岛素样生长因子-1受体IGF-1R、胰岛素受体IR、胰岛素受体底物IRS-1/2、蛋白激酶B PKB/Akt2、葡萄糖转运体4 GLUT4)、脂联素和瘦素信号通路(脂联素受体2 AdipoR2、a型瘦素受体OBRa、b型瘦素受体OBRb、AMP激酶α2 AMPKa2)的表达变化。ELISA试剂盒检测胎血脂联素水平。基于核磁共振(NMR)的代谢组学技术用于检测胎血中糖代谢、脂代谢和氨基酸代谢产物的变化。结果：①胰岛素抵抗相关代谢信号通路：咖啡因处理组可显著降低胎肝IGF-1、IGF-1R、IR的表达以及胎骨骼肌IR的表达(P<0.05,P<0.01),升高胎肝AdipoR2、OBRa、OBRb和AMPKa2的表达(P<0.05,P<0.01),但胎肝和骨骼肌中IRS-2、IRS-1、Akt2和GLUT4的表达变化不明显。ELISA结果显示,咖啡因处理组可显著升高胎血脂联素水平(P<0.05)；②NMR检测和分析结果显示,咖啡因处理组可引起IUGR胎儿内生的多种糖、氨基酸和脂肪代谢产物含量改变,主要表现为：胎血中α-葡萄糖、β-葡萄糖、胆固醇、酪氨酸、色氨酸和苯丙氨酸含量显著性升高(P<0.05),极低密度脂蛋白、TG、异亮氨酸和缬氨酸含量显著性降低(P<0.05)；③GC活化代谢：咖啡因处理组的胎肝和骨骼肌均出现11β-HSD-1、GR表达升高(P<0.05),但11β-HSD-2表达降低。结论：孕期咖啡因所致“母源性高GC”可能通过激活外周组织(肝脏和骨骼肌)的GC活化代谢和GR表达,一方面抑制IGF类胰岛素/胰岛素信号通路的功能发育,另一方面促进脂联素信号通路和瘦素信号通路功能增强,从而使胎鼠代谢编程发生改变,NMR技术也证实孕期咖啡因暴露可导致胎鼠内生的多种糖、脂肪和氨基酸代谢产物含量改变。这些变化将直接减慢胎儿的生长发育速度,引起IUGR发生。咖啡因孕期暴露所致胎鼠代谢编程改变将一直延续至出生后甚至成年,成为成年胰岛素抵抗和代谢紊乱的宫内起端。
第四部分11β-HSD-2甲基化修饰改变介导咖啡因所致胎海马GC活化代谢和GR高表达
目的：在细胞水平,以胎海马神经元为代表,通过研究咖啡因处理-11β-HSD-2启动子甲基化增加-11β-HSD-1/11β-HSD-2表达比增加-GC活化代谢增强及GR表达增加之间的内在联系,证实11β-HSD-2甲基化修饰改变介导咖啡因所致胎海马GC活化代谢和GR高表达,探讨咖啡因引起IUGR及其成年代谢紊乱发生的表观遗传机制。方法：建立原代胎海马神经元体外培养系统,以0-300μM咖啡因处理胎海马神经元24 h,或以300μM咖啡因处理0、6、12、24、48和72 h,实时定量RT-PCR和/或Western blot技术检测海马神经元GR、11β-HSD-1和11p-HSD-2的mRNA和蛋白表达；商业化启动子甲基化芯片筛选300μM咖啡因处理胎海马神经元24 h后全基因启动子区的甲基化情况,亚硫酸氢盐修饰后测序(BSP)技术检测和分析11β-HSD-2启动子区的甲基化频率。结果：①GC活化代谢：不同浓度咖啡因处理能显著降低11β-HSD-2的mRNA表达(P<0.05,P<0.01),升高11β-HSD-1的mRNA表达以及GR的mRNA和蛋白表达(P<0.05)。皆呈现良好的时间相关性和浓度依赖性。②11p-HSD-2的甲基化修饰：甲基化芯片筛选发现,咖啡因处理组发生甲基化修饰的基因共1430个,发生去甲基化修饰基因共2337个。其中,海马11β-HSD-2启动子区-358--77 bp区域发生了显著的高甲基化发生率,为正常对照组的2.51倍。进一步的BSP验证结果显示,咖啡因组11β-HSD-2启动子区-358--77区域总甲基化率较对照组显著增加(P<0.05),在-220、-214、-211、-193、-167、-152、-130、-111bp处的CpG位点甲基化发生率均出现显著升高(P<0.05,P<0.01)。结论：咖啡因可能通过增加胎海马11p-HSD-2启动子区总甲基化频率,降低11β-HSD-2表达,由此抑制局部GC灭活代谢而诱导11β-HSD-1表达增加,后者将诱导GR表达增加。咖啡因对胎海马11β-HSD-2启动子区甲基化修饰的直接改变,还可能同时存在于胎盘和其他胎儿组织(如胎肝、胎骨骼肌),可能是引起这些组织GC活化代谢及GR功能增强的重要原因,也是最终引起IUGR及其成年胰岛素抵抗的宫内发生起端。
孕期咖啡因暴露通过增加胎盘和多种胎组织(如胎海马、肝脏和骨骼肌)11β-HSD-2启动子区的总甲基化频率并降低其表达,增加这些组织11β-HSD-1/11β-HSD-2的表达比,由此削弱胎盘对母源性GC的屏障作用,引起胎儿母源性GC过暴露,并促使胎HPA轴发育异常和外周组织代谢编程改变。孕期咖啡因暴露所致胎儿HPA轴功能和代谢编程改变还将延续至出生后甚至成年,表现为成年后基础HPA轴功能降低、应激反应敏感性增加以及代谢紊乱的发生。
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 ONE
Insulin Resistance and Metabolic Disorders in Adult Caffeine Induced IUGR Rats
Objective: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 TWO
Prenatal caffeine exposure induced maternal GC overexposure to fetus and abnormality of fetal HPA axis development
Objective: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 THREE
Prenatal caffeine exposure induced the changes of metabolic pathways and metabolites in peripheral tissues of fetal rats
Objective: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 FOUR
11β-HSD-2 methylation mediated caffeine induced IUGR and adult insulin resistance
Objective: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.
CONCLUSIONS
Caffeine 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.
引文
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