【作者】 李后开；

【导师】 贾伟；

【作者基本信息】 上海交通大学 ， 药理学， 2008， 博士

【摘要】 肥胖是机体能量摄入大于消耗的一种慢性能量平衡失调的结果,表现为机体脂肪组织量过多或脂肪组织的比例过高。近几十年来,大量的研究集中在引发肥胖的遗传学机制,以及各种环境因素诱发肥胖的病理基础,并取得了积极的成果。肥胖易感(obesity-prone, OP)和肥胖抵抗(obesity-resistant, OR)现象是指同一种属实验动物在接受相同的饮食喂养后,动物之间出现的两种体重表型,即部分动物产生明显的肥胖,而另一些动物的体重增长比例相对较低。OP与OR这两种表型的产生是遗传和环境因素之间互作的结果。针对OP与OR大鼠的研究将会有助于进一步认识肥胖及其所诱发的代谢性异常发生的机制,为寻找肥胖发生规律及有效的防治措施提供实验依据。目前,未见采用代谢组学与转录组学对OP与OR大鼠之间的代谢与基因表达进行对比研究的报道。因此,本课题采用了代谢组学、转录组学与分子生物学方法对OP与OR大鼠之间的代谢与基因表达进行对比研究,主要实验内容和结果包括:⑴大鼠对高脂饮食喂养不同反应的药物代谢组学研究利用药物代谢组学(Pharmaco-metabonomics)方法,发现了大鼠高脂饮食喂养前的基础代谢表型存在差异,主要包括肠道菌群结构以及能量代谢上的差异等,这些基础代谢表型上的差异与大鼠对高脂饮食干预后体重差异相关。同时,在链尿佐菌素(STZ)诱导的大鼠糖尿病模型上进一步验证了药物代谢组学方法,并发现大鼠化学干预前的尿液代谢谱与其血糖升高水平相关。⑵肥胖易感与肥胖抵抗大鼠生化代谢特征的比较各种生化分析表明OP与OR大鼠在肝脏脂质代谢和胰岛素敏感性等方面表现出显著性差异。胰岛素耐受实验和胰岛素敏感指数(HOMA-IR)表明OP动物的胰岛素敏感性较OR动物下降,而OP大鼠血清中游离脂肪酸、酮体、肝脏总胆固醇和甘油三酯水平显著升高;但是,OP与OR大鼠血清中总胆固醇、甘油三酯、低密度脂蛋白、高密度脂蛋白和空腹血糖等的水平并无显著性差异;⑶肥胖易感与肥胖抵抗大鼠血清、尿液和肝脏组织提取物中代谢物的比较研究表明:①OP与OR大鼠的血清、尿液和肝组织提取物中多种氨基酸的含量存在显著差异,并以肝组织中的差异氨基酸数量为最多,包括各种生酮和生糖氨基酸水平在OP组的升高,说明氨基酸代谢的差异是两种体重表型大鼠之间存在的重要差异特征之一;②OP与OR动物肝脏和血清差异代谢物中包含多种饱和长链脂肪酸的升高如十四烷酸、十六烷酸、硬脂酸等和多不饱和脂肪酸的下降如亚油酸和花生四烯酸,说明两种体重表型动物的肝脏脂肪酸代谢存在明显差异;③长期高脂饮食喂养后,动物的尿液代谢物分析表明OP与OR动物体内的肠道菌群结构存在差异,这些菌群上的差别可能在动物体重增长的调节上产生影响;④与OR动物相比,OP动物尿液代谢物中儿茶酚胺类递质的代谢终产物如高香草酸、扁桃酸和4-羟基苯乙酸明显升高。同时,OP大鼠血清中三羧酸循环中间体柠檬酸、α-酮戊二酸和苹果酸等也显著高于OR大鼠,说明OP大鼠体内的交感神经系统活性及能量代谢过程更为活跃,可能与OP动物体内更多的能量需求有关。⑷肥胖易感与肥胖抵抗大鼠转录组学比较发现两组动物肝脏中有近80种基因在转录水平表现出显著差异(≥1.5倍),差异表达的基因主要涉及到脂质代谢、酮体生成、酶调节和转录调节等代谢通路。结合RT-PCR验证的结果,发现部分重要的基因如Ephx2, Nr3c2, Igfals, Pparg, Apoa4, Hmgcs1等在OP大鼠中明显上调。差异表达的基因及相关的代谢组学结果表明肥胖个体中易发代谢性异常的部分机制和相关的代谢途径。本课题通过对高脂饮食喂养和STZ干预前大鼠尿液的代谢物分析,发现大鼠高脂饮食或化学干预前尿液中代谢物谱存在差异,而这些差异与动物干预后体重增长或血糖升高出现不同反应密切相关,这些结果为未来采用代谢组学方法预测药理学结果提供了实验依据。高脂饮食喂养建立的OP大鼠体内的整体代谢特征与OR大鼠不同,具体包括:交感神经系统活性、能量代谢、脂肪酸代谢、氨基酸代谢以及肠道菌群结构等;OP和OR两种表型的转录组学比较也揭示了部分参与脂质代谢、酮体生成、血压调节和脂肪细胞分化等调控过程的基因存在差异性表达。两种组学研究手段的结合为系统地阐释肥胖个体的代谢特征及其发生机制提供了更加全面的信息,也为不同组学在复杂性疾病机制的整合研究提供了有益的参考和方法学。更多还原

【Abstract】 Obesity is the result of energy imbalance within an organism, that is, energy intake exceeds energy expenditure, characterized with excessive accumulation of fat mass or the higher proportion of adipose tissue to body weight. During the past decades, a large number of researches have been focused on the genetic etiology and the pathological basis of environmental factors of obesity. Obesity-prone (OP) and obesity-resistant (OR) phenomena are two body weight phenotypes among animals within the same strain which are fed with identical diet. The OP and OR phenotypes are interactive results of both genetic and environmental factors. As a result, the comprehensive study on OP and OR rats would facilitate the elucidation of mechanism of obesity, and the obese-related metabolic disorders. Currently, there is no report on comparison of metabolic and transcript profiles between OP and OR rats with metabonomic and transcriptomic approaches. Therefore, we intend to compare the metabolic and transcript profiles between OP and OR rats with the approaches of metabonomics, transcriptomics and molecular biology in this study. The main results are as following:⑴Pharmaco-metabonomic study on different responses to high fat diet feeding in rats We have detected characterized urinary metabolic profiles of OP and OR rats before high fat diet feeding. These differences between OP and OR rats included structure of gut microflora and energy metabolism, which were relative with body weight gain after high fat diet feeding. Additionally, we also validated the pharmaco-metabonomic concept in streptozotocin (STZ)-induced diabete model, which revealed that the predose unrinary metabolic profile was predictive for the increasing extent of postdose blood glucose.⑵The comparisons of biochemistry between OP and OR rats The differences between OP and OR rats included not only body weight, but lipids metabolism and insulin sensitivity as well, characterized with insulin resistance, increasing in serum free fatty acids and ketone body, and hepatic TC and TG in OP rats. However, no significant differences were observed in serum TG, TC, LDL, HDL and fasting glucose between OP and OR rats.⑶Comparisons of metabolites in serum, urine and liver tissue between OP and OR rats①There were significant differences in amino acids concentration between OP and OR rats,especially in liver tissue, such as high concentrations in ketogenic and glucogenic amino acids in OP rats, suggesting differences in amino acids metabolism;② The different metabolites between OP and OR rats included increasing of various saturated fatty acids and decreasing of polyunsaturated fatty acids in OP rats;③The urinary metabolites analysis indicated that different structure or metabolism of gut microflora might exist between the two phenotypes, which probably influenced the regulation of body weight gain;④The end-products of catecholamines in urine and intermediates of krebs cycle in serum in OP rats were all up-regulated, suggesting that the activity of sympatheic nervous system and energy metabolism was higher in OP rats than OR rats.⑷Transcriptomic comparisons between OP and OR rats Nearly 80 differently expressed hepatic genes were found between OP and OR rats (more than 1.5 fold ratio). These genes were involved in pathways of lipids metabolism, ketone body production, enzymatic and transcriptional regulation, most of which were consistent with results of metabonomic investigation. Combined with real time RT-PCR, we found that genes such as Ephx2, Nr3c2, Igfals, Pparg, Apoa4 and Hmgcs1were up-regulated in OP rats. The differentially expressed genes and metabolites partly explained the metabolic processes to metabolic disorders among obese subjects.Our study discloses the relationship between pre-dose metabolic profiles and body weight increasing (or blood glucose rising) through analysis of urine samples before high fat diet feeding (or STZ injection), which provides experimental evidence for application of metabonomics in pharmacological evaluation in the future. The metabonomic investigation indicates that the global metabolic patterns between OP and OR rats are different, including sympathetic nervous system, energy metabolism, fatty acid metabolism, amino acid metabolism and gut microflora. The comparative transcript profiles between OP and OR rats reveal some differentially expressed genes involved in lipids metabolism, ketone body production, blood pressure regulation and adipoctyes differentiation processes.The combined strategy of metabonomics and transcriptomics provides comprehensive information of metabolic characters of obesity and corresponding mechanisms to obese-related metabolic disorders from a systematic view, e.g. gut microflora-involved body weight regulation, Ephx2 gene in catecholamines metabolism pathway, and Pparg in hepatic lipids transport. Therefore, this study provides practical reference for integration of different“omics”in research of complex diseases.更多还原