Lkbl维持斑马鱼葡萄糖代谢平衡的功能研究

发布时间：2018-02-26 18:37

本文关键词： Lkb1 葡萄糖代谢糖酵解丙酮酸脱氢酶激酶pdk2 斑马鱼早期发育　出处：《中国农业大学》2016年博士论文　论文类型：学位论文

【摘要】：丝氨酸／苏氨酸蛋白激酶LKB1的编码基因是Peutz-Jeghers综合症的致病基因。LKB1不仅是一个重要的肿瘤抑制因子,而且在细胞生长、极性和细胞能量代谢等过程中具有重要的调节功能。但在生理条件下,LKB1如何在整体水平上调控代谢平衡以及相关的生化机制尚待阐明。本研究通过利用Tol2转座子介导的斑马鱼基因捕获突变体筛选,获得了斑马鱼lkbl突变体。由To12转座子酶介导含有EGFP编码序列的转座子元件插入到lkbl基因的第一个内含子中。该突变体因剪接获得新的融合转录本,该融合转录本仅包含lkbl的第一个外显子和EGFP的编码序列。同时,由于斑马鱼lkbl基因的翻译起始密码子位于其第二个外显子中,因而由这个融合转录本翻译获得的融合蛋白仅包含EGFP自身,导致突变体中内源lkbl的表达在转录水平和蛋白水平被显著阻断。我们发现,在正常喂养条件下,lkbl突变体幼鱼在受精后第7天开始出现死亡,在受精后第25天内全部死亡；但在不饲喂条件下,lkbl突变体幼鱼在受精后第7天开始死亡,并且死亡速率显著增加,在受精后第12天前全部死亡。受精后第7天的1kbl突变体幼鱼的肝脏颜色明显变深,而且肠道壁的褶皱明显减少,能量存储异常快速地消耗衰竭,表现出饥饿应激的表型。Lkbl缺失导致幼鱼早期死亡,表明斑马鱼lkbl对维持生存是必需的。我们将研究关注在受精后第5天之前的发育时期,在此阶段,斑马鱼胚胎发育依赖于卵黄为其提供能量和物质供给,没有饥饿应激刺激,因此可以保证在正常生理条件下研究Lkbl在维持整体能量代谢平衡中的功能。我们的实验进一步证明,Lkbl缺失会破坏突变体体内葡萄糖水平的动态平衡。与野生型对照相比,lkbl突变体在受精后36小时和第2天分别表现出较高的葡萄糖水平,而在受精后第4天和第5天突变体体内的葡萄糖水平却显著低于野生型。这些结果首次证明,先于卵黄未消耗完之前,Lkbl缺失已导致斑马鱼胚胎整体葡萄糖水平异常,提示在斑马鱼早期发育过程中,Lkbl对维持葡萄糖代谢平衡是至关重要的。进一步研究发现,Lkbl缺失导致糖异生过程中关键的限速酶磷酸烯醇丙酮酸羧激酶1(pepck1)和葡萄糖-6-磷酸酶(g6pase)的转录水平显著上调。为了探究Lkbl缺失造成斑马鱼体内葡萄糖代谢异常的内在机制,我们首先检测了葡萄糖代谢的下游产物,发现lkbl突变体中乳酸水平异常升高。进一步研究表明,Lkbl缺失导致丙酮酸脱氢酶激酶2(pdk2)的表达水平上调,进而造成丙酮酸脱氢酶(Pdh)磷酸化水平升高,并由此导致丙酮酸脱氢酶复合体(Pdc)活性下降,使得葡萄糖代谢向乳酸生成增多的糖酵解方向进行,造成lkbl突变体中乳酸水平异常升高。这可能是Lkbl缺失导致斑马鱼体内葡萄糖代谢平衡异常的内在的生化机制。由Lkbl缺失造成的葡萄糖代谢异常与肿瘤细胞所特有的有氧糖酵解增强,即Warburg效应高度相似。利用癌症治疗的候选药物二氯乙酸盐DCA (Dichloroacetate)抑制Pdk2活性,可以增强Pdc活性,有效地挽救由Lkbl缺失造成的乳酸积累异常的表型。提示我们的lkbl突变体或许可以为癌症治疗中以有氧糖酵解为靶点的药物筛选提供一个可能的平台。
[Abstract]:The gene encoding serine / threonine protein kinase LKB1 is a causative gene of.LKB1 Peutz-Jeghers syndrome is not only an important tumor suppressor, and in cell growth, has an important regulatory function of polarity and cell energy metabolism. But under physiological conditions, LKB1 in the overall level of metabolic balance and related regulation the biochemical mechanism remains to be elucidated. In this study, the zebrafish genes transposon mediated by Tol2 capture mutant selection, the zebrafish lkbl mutant. By To12 transposon mediated enzyme containing inserted into the first intron of the lkbl gene in transposable element EGFP encoding sequences. The mutant was obtained new fusion transcript splicing and the first exon and EGFP sequence encoding the fusion transcript contains only lkbl. At the same time, because of the translation initiation codon of lkbl gene in the zebrafish The second exons, and the fusion transcript translation fusion protein contains only the EGFP itself, leading to the endogenous expression of lkbl mutants was significantly inhibited at the transcriptional level and protein level. We found that in normal feeding conditions, the lkbl mutant in juvenile sperm appeared seventh days after the death, the death of all in twenty-fifth days after fertilization; but in feeding conditions, lkbl mutant juvenile began to die in seventh days after fertilization, and the death rate increased significantly in twelfth days after fertilization. After fertilization 1kbl mutant died seventh days juvenile liver color was darker, and the intestinal wall fold significantly reduced, energy storage abnormal fast consumption failure, showed the phenotype of.Lkbl deletion of hunger stress leads to early death of juvenile zebrafish lkbl, that is necessary for survival. We will focus on research after fertilization Fifth days before the period of development, at this stage, zebrafish embryo development depends on the yolk to provide energy and material supplies for its hunger, stress, so we can study Lkbl while maintaining the overall balance of energy metabolism in function in normal physiological conditions. We guarantee the experiment further proved that the dynamic balance of Lkbl deletion disrupts the glucose level the body of the mutant. Compared with wild-type controls, lkbl mutants in 36 hours after fertilization and second days respectively showed higher levels of glucose, and in fourth days and fifth days after fertilization in grape sugar levels but mutants were significantly lower than the wild type. These results demonstrate for the first time, before the yolk did not consume, Lkbl has been missing in zebrafish embryos whole abnormal glucose levels, suggesting that in the early development of zebrafish, Lkbl is vital to maintain glucose homeostasis further. The study found that Lkbl deletion resulted in gluconeogenesis in enzyme phosphoenolpyruvate carboxykinase 1 key (pepck1) and glucose -6- phosphatase (G6Pase) transcription level was significantly increased. In order to explore the internal mechanism of Lkbl caused by the lack of abnormal glucose metabolism in zebrafish, we first examined the downstream products of glucose metabolism and lactate level the lkbl mutant abnormalincreased. Further study showed that Lkbl deletion resulted in pyruvate dehydrogenase kinase 2 (PDK2) expression level increased, resulting in pyruvate dehydrogenase (Pdh) phosphorylation level increases, which leads to the pyruvate dehydrogenase complex (Pdc) activity decreased the glucose metabolism to lactate production increased glycolysis direction. The levels of lactic acid in the lkbl mutant caused by abnormal. This may be caused by Lkbl deletion in zebrafish glucose generation Xie Ping scale abnormal inner The biochemical mechanism of glucose metabolism caused by Lkbl deficiency. The abnormal and specific tumor cells of aerobic glycolysis is enhanced and that the Warburg effect is highly similar. Using the two drug candidate chloracetate DCA cancer treatment (Dichloroacetate) inhibit the activity of Pdk2, can enhance the activity of Pdc, effectively save the Lkbl caused by the lack of accumulation of lactic acid the abnormal phenotype. We suggest that lkbl mutants may be for cancer treatment by aerobic glycolysis for drug target screening may provide a platform.

【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q78;Q591.4

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