大鼠生后早期营养状况对成年期胰岛素抵抗效应的机制研究

发布时间：2018-07-04 10:17

本文选题：大鼠 + 营养过度　；参考：《上海交通大学》2015年博士论文

【摘要】：研究目的本研究通过生后早期营养干预致大鼠成年期胰岛素抵抗的动物模型来探讨生后早期营养环境与远期代谢效应的关联及其部分内在机制,阐明生后早期营养状况对成年期胰岛素抵抗的重要作用,为制定合理的生后早期喂养策略、预防和减少包括胰岛素抵抗在内的代谢综合症的发生提供依据。内容本研究将分成三部分。第一部分为表型确立,建立生后早期营养过度、营养正常和营养不足的大鼠模型,比较各组大鼠胰岛素抵抗差异。第二部分为机制探索,筛选与胰岛素抵抗相关的信号通路并测定其中重要信号因子在胰岛素效应器官的表达和表观遗传修饰作用,同时对血清代谢组学进行检测分析。第三部分为膳食刺激,比较生后早期不同营养状况大鼠在高脂饮食暴露下发生成年期IR的风险和重要信号因子表达的差异。方法新生SD大鼠,于生后第2天按每窝每只保姆鼠携带仔鼠数量的不同分别制备生后早期营养过度(3只/窝,SL组)、营养正常(10只/窝,NL组)和营养不足(20只/窝,LL组)大鼠模型。母鼠予常规饲料喂养。雄性大鼠为研究对象。仔鼠至日龄21天断乳后继续予常规饲料喂养。至6周龄时,每组再随机分成两个亚组,分别给予常规饲料和高脂饲料喂养至16周龄(成年期)止。动态观察各组大鼠生理生化指标、胰岛素抵抗情况和胰岛素外周效应器官(骨骼肌、内脏白色脂肪组织和肝脏)的形态学改变。运用转录组高通量测序技术筛选与胰岛素抵抗相关的信号通路,采用实时定量PCR和western blotting方法检测主要信号因子的表达,并使用重亚硫酸盐测序法对重要差异信号因子DNA甲基化的程度进行分析。此外,气相-质谱、超高液相色谱-质谱技术分别被用于大鼠血清脂肪酸谱和氨基酸谱的检测。结果1. SL组大鼠在3周龄和16周龄时体重分别较NL组大鼠增加了37.5%和15.1%,LL组大鼠体重分别降低了34.9%和12.6%。肝脏、骨骼肌和内脏白色脂肪(附睾脂肪、肾周脂肪)重量和组间差异与体重相-致,但经体重纠正后只有SL组与NL组内脏白色脂肪重量的组间差异得以保留。高脂喂养后仍然维持大鼠体重和内脏白色脂肪组织重量的组间差异。2.16周龄SL组大鼠出现胰岛素抵抗指数明显上升和葡萄糖不耐受,同时伴有血清甘油三酯和游离脂肪酸水平升高,但空腹血糖水平未受影响。LL组大鼠胰岛素抵抗指标与NL组相比没有差异。3. SL组大鼠3周龄时附睾脂肪细胞数目增加,16周龄时单个脂肪细胞面积增加。LL组大鼠单个附睾脂肪细胞面积均少于NL组大鼠。4.筛选出胰岛素信号通路作为研究的目标信号通路,其中主要关注的信号因子为胰岛素受体(Insr)、胰岛素受体底物(Irsl)、蛋白激酶B(Akt2)和葡萄糖转运体4(Glut4)。16周龄SL组大鼠附睾脂肪Glut4和Irs1的mRNA表达下调,Insr、Irs1、Akt2和Glut4的蛋白水平降低；腓肠肌Akt2和Glut4的mRNA表达下调、Insr和Glut4的蛋白水平下降。16周龄LL组大鼠附睾脂肪Glut4mRNA表达下调、腓肠肌Akt2mRNA表达上调和Irsl蛋白水平上升。5. 与NL组相比,SL组大鼠附睾脂肪Glut 4基因3周龄时为低甲基化高表达,16周龄时为高甲基化低表达。6.16周龄SL组大鼠血清长链饱和脂肪酸、单不饱和脂肪酸和花生四烯酸水平升高,同时伴有血清牛磺酸水平的降低。7. SL组大鼠高脂喂养后胰岛素抵抗指数和葡萄糖不耐受程度进一步上升,并出现明显的高瘦素血症和肝脏脂肪变性。附睾脂肪和腓肠肌胰岛素信号通路信号因子表达进一步下调。8. LL组大鼠高脂喂养后出现葡萄糖不耐受程度增加、内脏脂肪细胞面积明显扩大和显著的肝脏脂肪变性,同时伴有附睾脂肪和腓肠肌胰岛素信号通路部分信号因子表达明显下调。结论1. 生后早期营养过度可导致大鼠持续的体重过重和内脏白色脂肪堆积,生后早期营养不足与之相反,这种变化不受饮食因素的影响。2. 生后早期营养过度导致大鼠成年期IR。大鼠骨骼肌和内脏白色脂肪组织胰岛素信号通路受损、内脏白色脂肪组织Glut4基因甲基化程度改变、循环FFA水平增加、脂肪酸谱和氨基酸谱异常可能参与了IR的发生和进展。3. 高脂饮食加剧了生后早期营养过度大鼠的IR和胰岛素信号通路受损。4. 生后早期营养不足未对大鼠成年期IR产生影响,但高脂饮食可显著增加生后早期营养不足大鼠发生IR的风险。
[Abstract]:The purpose of this study is to explore the relationship between the early postnatal nutrition environment and the long-term metabolic effect and the intrinsic mechanism in order to elucidate the important role of early postnatal nutrition status to adult insulin resistance, and to formulate a reasonable early feeding strategy by using the animal model of adult insulin resistance induced by early postnatal nutrition intervention to explore the relationship between the early postnatal nutrition environment and the long-term metabolic effect. To prevent and reduce the occurrence of metabolic syndrome, including insulin resistance, this study will be divided into three parts. The first part is the establishment of phenotypes, the establishment of early postnatal overnutrition, normal nutrition and deficiency of the rat model, and the comparison of islet resistance differences in each group. The second part is the mechanism exploration, screening and analysis. Insulin resistance related signaling pathways and the determination of important signal factors in the expression of insulin effector organs and epigenetic modification, and the detection and analysis of serum metabolomics. The third part is dietary stimulation, and the risk of adult IR during the early stage of high fat diet exposure in rats with different nutritional status after birth is compared. Methods the difference in the expression of important signal factors. Method new SD rats were prepared on the second day after birth by each nanny rat of each nest to prepare early postnatal overnutrition (3 / nest, SL group), nutrition normal (10 / nest, NL group) and undernourishment (20 / LL) rats model. The female rats were fed with conventional feed. The male rats were fed. At the age of 21 days, the offspring continued to feed with conventional feed. At the age of 6 weeks, each group was randomly divided into two subgroups and fed with conventional and high fat feed to 16 weeks of age (adult). Dynamic observation of the physiological and biochemical indices of the rats, insulin resistance and the insulin peripheral effect organs (skeletal muscle, visceral white) Morphological changes in color adipose tissue and liver. The signal pathway related to insulin resistance was screened by high throughput sequencing technology in the transcriptional group. The expression of major signal factors was detected by real-time quantitative PCR and Western blotting methods, and the degree of DNA methylation of important differential signal factors was analyzed by heavy sulfite sequencing. In addition, GC-MS was used to detect the serum fatty acid spectrum and amino acid spectrum of rat serum. Results 1. SL rats were increased by 37.5% and 15.1% at the age of 3 and 16 weeks, respectively. The body weight of group LL decreased by 34.9% and 12.6%., and in the skeletal and visceral white fat respectively. The difference between the weight and the weight difference between the group SL and the NL group was retained after the weight correction. The difference between the weight of the body weight and the visceral white fat tissue after the high fat feeding was still maintained after the high fat feeding. The insulin resistance index in the group of SL rats at the age of.2.16 weeks was significantly higher. The level of triglyceride and free fatty acid in the serum was accompanied by elevated glucose intolerance and glucose intolerance, but the insulin resistance index of the.LL group was not significantly different from that of the NL group. The number of epididymal adipocytes in the group of.3. SL rats increased at the age of 3 weeks, and the area of the single adipocyte increased in the group of.LL rats at the age of 16 weeks. The area of the adipocyte was less than that of the NL group.4.. The insulin signaling pathway was selected as the target signal pathway for the study. The main signal factors concerned were insulin receptor (Insr), insulin receptor substrate (Irsl), protein kinase B (Akt2) and glucose transporter 4 (Glut4).16 weeks old rats in the epididymal fat Glut4 and Irs1 mRNA The protein levels of Insr, Irs1, Akt2 and Glut4 decreased, the expression of the mRNA expression of the gastrocnemius Akt2 and Glut4 was down, the protein levels of Insr and Glut4 decreased in the LL group of.16 weeks old rats, and the expression of the epididymal expression of the gastrocnemius muscle and the level of protein increased at the age of 3 weeks of age of the epididymal fat 4 of the rats. The high expression of low methylation was high methylation at the age of.6.16 week old SL group, the level of serum long chain saturated fatty acid, monounsaturated fatty acid and arachidic acid increased, and the level of serum taurine decreased in group.7. SL, and the index of insulin resistance and the degree of glucose intolerance increased further after high fat feeding in group.7. SL. The expression of signal factor of epididymal fat and gastrocnemius insulin signaling pathway further downregulated the increase of glucose intolerance after high fat feeding in the.8. LL group, significantly increased visceral adipocyte area and significant hepatic steatosis, accompanied by epididymal fat and gastrocnemius. The expression of partial signal factors in the signaling pathway of muscle insulin was obviously down-regulated. Conclusion early nutrition over 1. birth could lead to excessive weight loss and visceral white fat accumulation in rats. The early nutritional deficiency after birth was the opposite. This change was not influenced by dietary factors. The early period of.2. after birth was caused by the skeletal muscle of IR. rats in adult rats. The insulin signaling pathway in muscle and visceral white adipose tissue is damaged, the degree of methylation of Glut4 gene in visceral white adipose tissue changes, the level of circulating FFA increases, the abnormal fatty acid spectrum and amino acid spectrum may participate in the occurrence and progression of IR, and.3. high fat diet aggravates the IR and insulin signaling pathway of.4. in early postnatal rats. Early postnatal undernutrition did not affect IR in adult rats, but high fat diet significantly increased the risk of IR in the early postnatal undernourished rats.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R153.1

【相似文献】