【作者】 刘晓梅；

【导师】 李书琴；

【作者基本信息】 中国医科大学 ， 儿科学， 2007， 博士

【摘要】 目的胎儿宫内发育迟缓（IUGR），又称为胎儿生长受限（FGR），是围产期主要并发症之一，其发病率在不同国家与地区不尽一致，我国IUGR发生率为6.39-16.3％。IUGR儿由于机体胰岛素抵抗导致成年期代谢综合征（包括肥胖、高血压、冠心病、糖耐量异常和2型糖尿病等）的发病率明显增高。IUGR个体发生胰岛素抵抗的机制尚不清楚，目前认为孕期营养不良可能导致子代某些器官或组织的结构或功能发生永久性改变。Hales和Barker提出的“节俭表型假说”得到普遍赞同，该假说认为不良的子宫内环境引起胎儿胰岛B细胞数量减少或功能异常；为保证重要脏器如脑的发育，外周组织（肝脏、脂肪、骨骼肌等）的发育和代谢类型发生“永久性”改变，产生胰岛素抵抗。胰岛素抵抗是指机体内胰岛素与其特异性受体结合后生物效应低于正常，它与胰岛素信号转导的各个环节、调控糖脂代谢的多种基因的分子异常和多态性相关。PI-3K途径是胰岛素代谢作用主要信号转导途径之一。胰岛素受体激活后，使胰岛素受体底物（IRS）磷酸化，磷酸化的IRS1／2激活PI-3K的p110亚单位，通过一系列级联反应活化PDK再激活蛋白激酶B（PKB），肝脏中PKBt激活后使糖原合成酶激酶（GSK）3丝氨酸残基磷酸化而失活，加速糖原正常合成。糖异生增加导致的肝葡萄糖输出增多是2型糖尿病机体胰岛素抵抗的重要组成部分。肝脏糖异生的关键调节因子是辅激活因子过氧化物酶体增殖因子活化受体γ的辅激活因子（PGC-1）α，PGC-1α对糖异生的调控主要发生在转录水平，可诱导肝细胞糖异生关键酶组，包括磷酸烯醇丙酮酸羧激酶（PEPCK）、葡萄糖-6-磷酸酶（G6Pase）和果糖1，6-二磷酸酶（FBPase），从而引起肝糖输出增加。蛋白质和氨基酸的缺乏是慢性营养不良的关键因素，我们采用孕期全程蛋白营养不良法建立大鼠IUGR模型，观察IUGR鼠的出生体重和不同发育阶段的体重、肾周脂肪重量和脏器重量，检测血清和肝脏中的糖原和游离脂肪酸的含量以及血糖和胰岛素水平，通过胰岛素耐量实验和基础状态法，了解母鼠孕期营养不良所致IUGR雄鼠在发育的不同阶段体内胰岛素敏感性的变化。检测肝脏中胰岛素信号传导和效应分子IRS-2、PI-3Kp110、PKB、GSK-3β等的蛋白表达和磷酸化水平，同时检测肝脏中转录激活因子PGC-1α的蛋白表达变化，糖异生关键酶PEPCK和G6Pase的mRNA表达变化。通过比较各组成年子鼠肝脏胰岛素信号分子的变化，以及不同发育阶段肝脏糖异生酶差异，分析孕期低蛋白与机体胰岛素抵抗发生的关系，探讨IUGR儿成年后发生胰岛素抵抗的分子机制。实验方法一、动物模型的制备健康Wistar大鼠，体重230-280g，雌性均为处女鼠，大鼠按雌：雄4：1合笼。孕鼠按受孕顺序随机分成IUGR组和正常对照组，对照组孕鼠饲以标准饲料（热卡为1583KJ／100g，蛋白质含量为23％），IUGR组孕鼠自妊娠第1天饲以低蛋白饲料（热卡为1558KJ／100g，蛋白质含量为8％）。所有孕鼠自然分娩，称量12h内新生子鼠体重，精确到0.01g。IUGR诊断标准：新生鼠出生体重低于正常对照组出生平均体重的2个标准差的为IUGR鼠二、实验对象选择分娩后母乳喂养，两组母鼠都饲以标准饲料。IUGR组和正常对照组（CON）幼鼠满3周后断乳，分笼饲以标准饲料。1、幼鼠标本采集及处理满3周龄时每组随机选6只雄性子鼠禁食12h后采用摘眼球法取血，静置15min后，3000rpm离心10min，血清于-70℃保存。取血后立即处死，测量肝脏、肾脏、心脏、全脑重量，精确到0.001g，标本于-70℃保存。2、成年鼠标本采集及处理满8周龄后，IUGR组和正常对照组雄性子鼠各随机取6只空腹12小时后称量体重，由尾静脉取血，静置15min后，3000rpm离心10min，血清-70℃保存，取脑、心脏、肝脏、肾脏、胰腺、和肾周脂肪标本并称重，精确到0．001g，标本于-70℃保存。3、成年鼠胰岛素耐量实验8周龄雄性子鼠每组随机选6只空腹12小时后称量体重，乌拉坦腹腔注射麻醉，分离右下肢股静脉插入18G套管针留置，腹腔注射胰岛素溶液0.5U／kg，在0min、15min、30min、60min每次抽取0.2ml血，血清-70℃冻存。三、实验方法1、测量新生鼠出生体重和1到8周不同发育阶段的体重，测量3周和8周子鼠的全脑、心脏、肝脏、肾脏、胰腺、和肾周脂肪重量。2、生化方法检测3周和8周子鼠血清及肝脏中游离脂肪酸和糖原的含量。3、采用葡萄糖氧化酶法检测3周和8周子鼠空腹血糖浓度和胰岛素耐量实验中各个时间点的血糖值。4、采用ELISA方法检测3周和8周子鼠空腹血清胰岛素水平。5、PGC-1α、PEPCK和G6Pase表达的检测：采用Western杂交检测3周和8周子鼠肝脏组织中PGC-1α的蛋白表达，采用RT-PCR法检测PGC-1α、PEPCK和G6Pase的mRNA水平。6、PI-3K途径分子表达的检测：Western杂交检测8周子鼠肝脏组织胰岛素信号传导和效应分子IRS-2、PI-3K、PKB以及GSK-3β的蛋白表达，同时测定PKB在胰岛素刺激前后的磷酸化水平变化。实验结果一、孕期蛋白质缺乏对子鼠生长发育的影响IUGR组IUGR发生率明显高于正常对照组（P＜0.001），IUGR组子鼠的平均出生体重比对照组比低约40％（P＜0.001）。满4周时IUGR组平均体重达到对照组平均水平，8周时IUGR组体重超过正常对照组（P＜0.05）。3周龄IUGR组幼鼠器官重量虽然普遍略低于正常对照，但没有统计学意义，只有肾脏重量IUGR组与对照组的差异有统计学意义（P＜0.05），8周时肾周脂肪重量IUGR组明显高于对照组（P＜0.01）。二、IUGR子鼠糖脂代谢和胰岛素敏感性的改变3周龄幼鼠的空腹血糖和胰岛素浓度以及IRI比较无明显差别。肝脏中FFA浓度和糖原含量两组之间差异也无统计学意义。8周IUGR子鼠空腹血清胰岛素浓度升高（P＜0.01），IRI比较有显著性差异（P＜0.05），肝脏FFA浓度和糖原含量差异无统计学意义，血清FFA浓度IUGR组高于对照组（P＜0.05）。胰岛素耐量实验中可见IUGR鼠对外源性胰岛素反应迟钝，胰岛素刺激15min后血糖较基础值仅降低25％，而对照组降低44％；2组在30min时血糖均降低至最低值，IUGR组血糖降低31％，对照组降低54％；60min时血糖己上升，IUGR组和对照组血糖较基础值降低分别是19％和34％。胰岛素刺激后IUGR组各个时间点血糖较基础值降低的幅度都明显低于对照组。三、IUGR子鼠肝脏PI-3K途径分子表达变化1、肝脏IRS-2、PI-3K和GSK-3β的蛋白表达变化Western blot检测结果显示，8周时IUGR组大鼠肝脏IRS-2的蛋白表达与正常对照没有差别（P=0.371）；PI-3K的催化亚单位p110的蛋白表达明显降低（P＜0.01）；而IUGR组大鼠肝脏中GSK-3β蛋白表达增加（P＜0.01）。2、肝脏PKB的蛋白表达及磷酸化水平的变化与对照组相比，在基础状态和胰岛素刺激状态下，8周IUGR组大鼠肝脏PKB蛋白和磷酸化的PKB^Ser473表达都明显降低（P＜0.01），胰岛素刺激状态下，对照组肝脏磷酸化的PKB^Ser473表达明显增加，是基础状态下的182％（P＜0.01），而IUGR组肝脏磷酸化的PKB^Ser473的增加不明显，仅是基础状态下的123％（P=0.024）。四、IUGR子鼠肝脏PGC-1α和糖异生酶表达的变化1、肝脏PGC-1α表达水平的变化RT-PCR结果显示，IUGR组3周幼鼠（P＜0.01）和8周成年鼠（P＜0.05）肝脏PGC-1α的mRNA相对表达量明显高于正常对照；Western blot结果也证实，在3周和8周，IUGR鼠肝脏中PGC-1α的蛋白水平都明显增加，分别是对照组的162％和230％（P＜0.05）。2、肝脏PEPCK和G6PasemRNA水平的改变采用RT-PCR技术检测发现，3周IUGR组肝脏PEPCK的mRNA的表达略高于正常对照（P＜0.05），8周时IUGR组的PEPCK相对表达量显著高于正常对照（P＜0.01）。无论幼鼠还是成年鼠，肝脏中G6Pase的mRNA表达水平，IUGR组都明显高于正常对照（P＜0.01）。而且PEPCK（r=0.907，P＜0.01）和G6Pase（r=0.726，P＜0.01）的mRNA水平与PGC-1的mRNA含量显示高度正相关。结论1、宫内蛋白营养不良可导致大鼠IUGR，IUGR子鼠生后出现生长追赶，成年后较正常同龄鼠肥胖，肾周脂肪沉积，出现糖脂代谢异常。2、IUGR子鼠幼年血糖和胰岛素水平与对照没有差异，成年后出现高胰岛素血症，IRI增高，胰岛素耐量实验证实IUGR鼠体内存在胰岛素抵抗。3、IUGR成年子鼠肝脏PI-3K蛋白表达降低，PKB的蛋白表达和磷酸化水平都降低，胰岛素刺激后表达增加的不明显，说明肝脏胰岛素信号通路PI-3K／PKB途径分子异常，对胰岛素反应敏感性降低。4、IUGR成年子鼠肝脏中PI-3K／PKB途径分子异常，导致GSK-3的表达增加，可能造成对肝脏糖原合成酶的抑制增强，促进肝葡萄糖输出，构成IUGR大鼠肝脏胰岛素抵抗的机制。5、肝脏中PKB的蛋白表达和磷酸化水平降低，引起IUGR鼠自幼年至成年肝脏中糖异生的关键调控因子PGC-1α的mRNA和蛋白表达增加，诱导糖异生关键酶PEPCK和G6Pase的mRNA表达增加，促进糖异生，增加肝脏葡萄糖输出，促进高血糖的发生。更多还原

【Abstract】 Intrauterine growth retardation (IUGR), also called fetal growth restriction (FGR), is a significant complication of pregnancy. In addition to perinatal morbidity and mortality, epidemiological studies suggest that IUGR may contribute to adverse health effects in adulthood including obesity, cardiovascular disease, impaired glucose tolerance, type 2diabetes, hypertension and other complications. Although the biochemical bases of these deleterious consequences are not well defined, it has been indicated that a poor nutrition during gestation results in metabolic changes in the offspring which can lead to permanent alterations in the structure and/or function of certain organs and tissues. The "thrifty phenotype hypothesis" proposed by Hales and Barker postulates that these changes occur in order to limit the use of nutrients by certain tissues, to ensure sufficient supply of them to the brain and other vital organs . Insulin resistance means the biological effect decreased after insulin combined with its specific receptor. It associated with abnormity and polymorphism of signal transduction and effecter molecule. Dysfunction of genes which regulate glucose and lipid metabolism can also cause insulin resistance. PI-3K pathway is the most principal way for insulin metabolism effect. Insulin combined and activated insulin receptor, subsequently phosphorylated insulin receptor substrates (IRS). Phosphorylated IRS activated phosphatidyliphosphatidylinositol 3-kinase (PI3-kinase). Through insulin-signaling cascade the phosphoinositide-dependent kinase-1 (PDK1) activated protein kinase B (PKB) . Phosphorylated PKB can made glycogen synthase kinase (GSK) 3 serine residue phosphorylation and deactivation subsequently accelerate glycogen synthesis. An important component of the peripheral insulin resistance associated with type 2 diabetes is impaired suppression of endogenous hepatic glucose production, which is predominately the result of gluconeogenesis. The enzymes glucose-6-phosphatase (G6Pase), phosphoenolpyruvate carboxykinase (PEPCK), and fructose-1, 6-bisphosphatase (FBPase) determine the rate of gluconeogenesis. In gluconeogenesis, the regulation of the rate-limiting (PEPCK) and the last committed (G6Pase) enzymes occurs mainly at the transcription level. It has been recently demonstrated that hepatic gene expression of these enzymes is controlled by peroxisome proliferator-activated receptor-γcoactivator-1 (PGC-1).Protein and amino acids deficiency are critical factors in chronic malnutrition. We established IUGR rat model by protein malnutrition during pregnancy. The incidence of IUGR and average birth weight were calculated. We observed the body weight, organ weight and perinephric fat pads at different weeks. Fasted serum glucose was determined by glucose oxidase method. Fasted serum insulin concentration were measured by enzyme-linked immunoabsordent assay (ELISA).in addition, glycogen and free fatty acid(FFA) of serum and hepatic were measured to study glucose lipid metabolism in IUGR rats.At 8 weeks of age, male rats were subjected to an insulin tolerance test (ITT), to evaluate insulin sensitivity in IUGR animals. Western blot analysis were undertaken to assess hepatic expression of IRS-2,PI-3K,PKB, phosphorylated Ser473-PKB protein and GSK-3 .the hepatic mRNA level of PEPCK and G6Pase and that of transcription factor PGC-1 which promote glucogenesis were determined . The present studies were performed to reveal the underlying mechanism responsible for insulin resistance in IUGR rats.Materials and Methods一、Animal modelHealthy Wistar rats (weight: 230-280g) .Female and male rats were raised in same cages by the proportion of four ratio one. Female rats were randomly divided into 2 groups: control group, IUGR group. Control maternal rats were fed with standard rat chow(caloriel583KJ/100g, protein 23%);animals of IUGR group were fed with low protein rat chow(calorie1558KJ/100g, protein 8%) since 1day of gestation. All the maternal rats were allowed to deliver spontaneously. Neonate rats were weighed on a scale with an accuracy of 0.01g.二、Experiment objectsJuvenal rats were weaned at 3 weeks of age, and then all fed with standard rat chow.1、Sample collection and treatmentOn 3 week and 8 week, after fasted for 12 hour, 0.2～0.5ml blood samples were collected from rats of each group. Blood samples were placed for 15 min and centrifuged at 3000 rpm for 10 minutes. The serum was stored at -70℃for later detection. The brain, heart, liver, kidney, pancreas and perinephric fat pads were harvested and weighted on a scale with an accuracy of 0.001g, quickly snap-frozen in lipid nitrogen and stored at -70℃.2、ITTOn 8 week, after fasted for 12 hour, in certain adult male animals, jugular vein catheters were placed surgically after intraperitoneal injection of ethyl carbamate. The animals received 0.5U/kg human insulin via intraperitoneal injection, and blood was obtained at 0, 15, 30, and 60 min subsequently to measure glucose concentration.三、Experiment Methods1、The body weights of rats since birth to 8 weeks of age were weighted. The weight of brain, heart, liver, kidney, pancreas and perinephric fat pads at W3 and W8 were examined. The diagnostic standard of IUGR is generally assigned to the Neonate rats measured with two standard deviation lower than the average birth weight of control group.2、The measurement of glycogen and FFA in the serum and hepatic: biochemical methods. 3、Serum glucose was determined by glucose oxidase method.4、Fasted serum insulin concentration was measured by ELISA.5、The measurement of PGC-1α、PEPCK and G6Pase mRNA levels in the hepatic: RT-PCR. The measurement of PGC-1αcprotein levels in the hepatic: Western blot.6、The measurement of IRS-2,PI-3K,PKB ,phosphorylated Ser473-PKB and GSK-3βprotein levels in the hepatic: Western blotResults一、The effects of protein deficiency during of pregnancy ongrowth and developmentThe incidence of IUGR in model group were significantly higher than control groups (P<0.01) . The average birth weight of IUGR group were significantly lower than that of control groups (P<0.05), until 4 weeks of age, when IUGR rats caught up to controls. Between 4～7 weeks of age, the growth of IUGR rats accelerated and surpassed that of controls at 8 weeks of age (P<0.05). IUGR rats were obese at 8 weeks of age with perinephric fat pads significantly increased compared with control rats (P<0.01) .二、Abnormal metabolism and insulin sensitivity in IUGR ratsNo significant differences were observed in blood glucose and insulin concentration at 3 week. Nor was the insulin resistance index (IRL). Hepatic concentration of FFA and glycogen did not differ at 3 week and 8 week between IUGR and control. But serum FFA concentration significantly increased compared with control. By 8 week IUGR rats developed hyperinsulinemia and high IRI (P<0.05) . Insulin tolerance test showed a significantly blunted glycemic response to exogenous insulin in IUGR rats (P<0.05) .三、Changes of PI-3K pathways signaling molecules in hepatic of adult IUGR rats 1、Hepatic protein levels of IRS-2、PI-3K and GSK-3βBasal levels of IRS-2 protein did not differ between IUGR and control rats (P=0.371) . The expression of PI-3K protein are decreased in adult IUGR rats compared with control (P<0.01) .whereas GSK-3βprotein level was significantly increased in IUGR rats (P<0.01) .2、Hepatic protein levels of PKB and phosphorylated PKBBoth PKB and phosphorylated Ser473-PKB protein levels are significantly decreased in liver of IUGR animals compared with controls (P<0.01) . After administration of insulin, phosphorylation at Ser473 of PKB significantly increased to 182% of basal level in control liver (P<0.01) ; However, phosphorylation of PKB in responded to insulin was markedly blunted in IUGR rats compared with controls, only increased to 123% of basal level (P=0.024) .四、Variation of PGC-1α,PEPCK and G6Pase in hepatic of IUGRrats1、Hepatic protein and mRNA levels of PGC-1αUsing the traditional RT-PCR method, we found that W 3 and W 8 hepatic mRNA levels of PGC-1 were significantly increased in IUGR livers compared with control values (P<0.05) . Hepatic protein levels of PGC-1 at W 3 and W 8 were significantly increased to 162 % and 230%of control values, respectively (P < 0.05)2、Hepatic mRNA levels of PEPCK and G6PaseHepatic mRNA levels of G6Pase at W 3 and W 8 were significantly increased in the IUGR animals (P<0.01) .Slimily, hepatic mRNA levels of PEPCK at W 3 and W 8 were increased in the IUGR animals compared with control rats. Moreover, when the extent of these increases was analyzed in comparison with the rise in PGC-1 mRNA levels on a sample-to-sample basis, we found that the G6Pase (r =0.726, P<0.01) , PEPCK (r =0.907, P<0.01) mRNA levels correlated directly to PGC-1 mRNA levels. Conclusions1、Exposed to protein malnutrition during pregnancy can induce IUGR. The IUGR rats catch up growth after birth, and to be fatter than normal rats when grown up. They appear to have more fatty deposit in internal organs and abnormal glucose and lipid metabolism2、No significant differences were observed in blood glucose and plasma insulin levels at 3 week of age. However, at age 8weeks, IUGR rats developed hyperinsulinemia and high IRI. Insulin tolerance test confirmed that IUGR rats developed insulin resistance.3、The expression of PI-3K protein are decreased in livers of adult IUGR rats. Both PKB and phosphorylated Ser473-PKB protein levels are significantly decreased in hepatic of IUGR animals compared with controls. After administration of insulin, phosphorylation of PKB in responded to insulin was markedly blunted in IUGR rats compared with controls. These data suggest there are postreceptor defect in the intracellular insulin -signaling cascade in IUGR animals.4、The expression levels of GSK-3P protein are increased in livers of adult IUGR rats. It may inhibit the activity of glycogen synthase and subsequent promote the glucose production.5、Hepatic protein and mRNA levels of PGC-1αare significantly increased in IUGR rats. It contributed to the increased mRNA levels of gluconeogenic enzymes PEPCK and G6Pase, and subsequently promotes hepatic gluconeogenesis. These changes may hasten the development of hyperglycaemia.更多还原