【作者】 陈艳；

【导师】 吴江维；

【作者基本信息】 西北农林科技大学 ， 动物遗传育种与繁殖， 2020， 硕士

【摘要】 线粒体对于维持白色脂肪细胞代谢稳态是必需的,在脂肪形成的初始步骤中,TCA循环产生的ATP维持了正常的脂质合成。在TCA循环中线粒体顺乌头酸酶(Mitochondrial aconitase,Aco2)负责催化柠檬酸到异柠檬酸的转化,在此过程中产生的NADH和FADH2,通过氧化磷酸化(Oxidative phosphorylation,OXPHOS)合成ATP。在这项研究中,我们通过过表达、缺失Aco2的方法探讨了脂质生成与线粒体能量代谢之间的关系,该研究结果有助于进一步了解TCA循环酶和线粒体功能与脂肪组织脂质代谢之间的关系,以期为肥胖及其相关代谢疾病患者的治疗提供一种潜在方法和为改善猪肉品质提供理论基础。本研究主要获得以下结果:1.Aco2在小鼠脂肪组织和3T3-L1脂肪细胞分化过程中的表达模式。高脂饮食(High fat diet-fed,HFD)小鼠腹股沟白色脂肪组织(Inguinal white adipose tissue,i WAT)、附睾白色脂肪组织(Epididymal white adipose tissue,e WAT)、棕色脂肪组织(Brown adipose tissue,BAT)和肝脏中Aco2的m RNA水平显著低于正常饮食(Chow diet-fed,CD)小鼠,但i WAT中Aco2酶的活性显著高于CD小鼠;4℃寒冷刺激下小鼠i WAT中Aco2 m RNA水平和酶活性均高于室温下小鼠;在3T3-L1脂肪细胞分化过程中,Aco2 m RNA水平和酶活性从第0天到第4天的早期分化阶段稳定表达,但在第4天到第8天的后期分化阶段显著升高。2.Aco2过表达促进3T3-L1脂肪细胞脂质生成。Aco2过表达在不影响细胞增殖的情况下显著增加了3T3-L1脂肪细胞内甘油三酯的含量,促进了脂质合成相关基因Acc、Fas和甘油三酯(Triglyceride,TG)合成相关基因Glut4、Ppar-γ、Dgat2的m RNA和蛋白表达水平,降低了HSL和ATGL两个脂质水解标志物的蛋白表达。3.Aco2过表达增强线粒体ATP产生。Aco2过表达通过增加脂肪细胞中线粒体编码基因表达、线粒体数量、线粒体DNA拷贝数和线粒体生成调节因子显著促进了线粒体生物合成;通过增加TCA循环限速酶柠檬酸合成酶活性,NADPH/NADP~+比例增强线粒体TCA循环速率;通过增加线粒体氧化磷酸化复合物蛋白表达提高了脂肪细胞ATP含量,证明了Aco2过表达促进了3T3-L1脂肪细胞中线粒体生成和ATP产生。为了进一步研究过表达Aco2引起的脂质生成增加归因于ATP产生的增加,在Aco2过表达的细胞中添加20μm鱼藤酮消耗细胞中ATP含量,发现添加了鱼藤酮的Aco2过表达细胞中脂质生成的促进作用被消除,表明ATP是Aco2过表达促进脂肪细胞脂质合成的限制因子。4.Aco2缺失抑制3T3-L1脂肪细胞脂质生成。在3T3-L1脂肪细胞中使用CRISPR/Cas9技术缺失Aco2后显示:Aco2缺失在不影响细胞增殖的情况下显著减少了3T3-L1脂肪细胞内甘油三酯含量、降低了脂质合成相关基因和TG合成相关基因的m RNA和蛋白表达水平,增加了脂质水解相关基因蛋白表达以及减少了TCA循环中间体柠檬酸、异柠檬酸、延胡索酸和苹果酸含量;为了进一步研究Aco2缺失抑制脂质合成的分子机制,我们检测了Aco2缺失和对照细胞中线粒体生成、TCA循环速率和胞内ATP含量,发现Aco2缺失抑制了3T3-L1脂肪细胞中线粒体生成和ATP产生。5.添加异柠檬酸恢复了Aco2缺失细胞的脂质合成。TCA循环中Aco2负责将柠檬酸转化为异柠檬酸,在Aco2缺失细胞中检测到异柠檬酸水平降低,接下来我们探究了在Aco2缺失细胞中外源添加异柠檬酸是否会恢复Aco2缺失细胞的脂质合成能力。通过向Aco2缺失细胞中添加5m M异柠檬酸,形态学上发现脂滴积累得到了恢复,脂质生成和TG合成相关基因的m RNA和蛋白质水平也得到提高,同时线粒体生成和ATP含量也得到了有效恢复,这些结果说明外源添加异柠檬酸盐可恢复Aco2缺失细胞的脂肪细胞分化;为了进一步测试ATP是否是异柠檬酸刺激脂肪生成的限制因素,我们向异柠檬酸处理过的Aco2缺失细胞中添加了20μm鱼藤酮,检测发现ATP的消耗完全废除了异柠檬酸盐对Aco2缺失细胞脂肪形成的影响,说明ATP也是异柠檬酸恢复Aco2缺失细胞脂质合成的限制因子。综上所述,Aco2对于3T3-L1脂肪细胞分化至关重要,Aco2过表达促进了3T3-L1脂肪细胞的脂质蓄积,Aco2缺失抑制了脂肪细胞的脂肪形成,添加异柠檬酸可以恢复Aco2缺失细胞的脂质合成;另一方面,机制上,Aco2在脂质生成中的促进作用是ATP依赖性的,通过阻止ATP产生,可以消除Aco2对脂质生成的影响。更多还原

【Abstract】 Mitochondria are necessary for maintaining white adipocyte metabolic homeostasis,TCA cycle produced ATP sustains normal lipid synthesis during the early period of adipogenesis.In the TCA cycle,mitochondrial aconitase(Aco2)catalyzes the conversion of citrate to isocitrate,which produces NADH and FADH2 driving synthesis of ATP through OXPHOS.In this study,we explore the relationship between adipogenesis and mitochondrial energy metabolism through overexpression and deficiency of Aco2.Our work is helpful to further understand the relationship between TCA cycle enzymes and mitochondrial function and lipid metabolism of adipose tissue,providing a potential method for treatment obesity and related metabolic syndrome and providing a theoretical basis for improving meat quality.The main results of this study are as follows:1.Expression patterns of Aco2 in adipose tissue of mice and during adipocyte differentiation in 3T3-L1 cells.Compared to CD mice,m RNA levels of Aco2 was significantly lower in i WAT,e WAT,BAT and livers of HFD mice.However,Aco2 enzyme activity in i WAT showing higher levels in HFD mice than that of CD mice.Levels of Aco2 m RNA and activity in i WAT of mice exposed at 4℃significantly higher than those at room temperature.During3T3-L1 cells differentiation,levels of Aco2 m RNA and activity showing stable expressions in the early differentiation period from day 0 to day 4,with increased levels in the late differentiation phase from day 4 to day 8.2.Overexpression of Aco2 promotes adipogenesis in 3T3-L1 cells.Overexpression of Aco2 promote triglyceride synthesis,increased m RNA and protein levels of lipogenesis Acc,Fas and TG synthesis Glut4,Ppar-γ,Dgat2 and reduced protein levels of two major adipose tissue lipases ATGL and HSL without affecting cell proliferation.3.Aco2 overexpression enhances mitochondrial ATP production.Aco2 overexpression significantly enhances mitochondrial biogenesis by increasing the m RNA expression of mitochondrially encoded genes,mitochondrial mass,mitochondrial DNA copy number and the m RNA levels of mitochondrial biogenesis regulators in adipocytes.By increasing the activity of citrate synthase(Cs),the ratio of NADPH/NADP~+enhances the rate of mitochondrial TCA cycling.By increasing the protein expression of select subunits of the OXPHOS complexes promoted the ATP level.Thus,proving that overexpression of Aco2enhances mitochondrial biogenesis and ATP synthesis in 3T3-L1 cells.Furthermore,in order to test that increased adipogenesis in Aco2-overexpressing cells was attributed to elevated ATP production.By added 20μm rotenone to Aco2-overexpressing cells to consume the ATP content in the cells,we found the promoting effect of Aco2 overexpressing on adipogenesis was abolished by rotenone treatment,suggesting that Aco2 overexpression mediates adipogenesis through elevation of ATP production4.Deficiency of Aco2 inhibits adipocytes differentiation.CRISPR/Cas9-mediated Aco2deficiency was applied in 3T3-L1 cells,the results show that deficiency of Aco2 inhibit triglyceride synthesis,reduced m RNA and protein levels of lipogenesis and TG synthesis,increased protein levels of two major adipose tissue lipases ATGL and HSL and show lower levels of citrate,isocitrate,fumarate as well as malate without affecting cell proliferation.Further,in order to study the mechanism of Aco2 deficiency inhibiting lipid synthesis,we examined the expression levels of mitochondrial biogenesis,TCA cycle rate and ATP content in Aco2 deficiency and control cells,these results demonstrate that deficiency of Aco2 reduces mitochondrial biogenesis and ATP synthesis in 3T3-L1 cells.5.Supplementation of isocitrate restores adipocyte differentiation in Aco2-deficient cells.Considering the role of Aco2 in the conversion of citrate to isocitrate in the TCA cycle,and we found that the level of isocitrate is reduced in Aco2-deficient cells.Next,we explored whether the supplementation of isocitrate restores adipocyte differentiation in Aco2-deficient cells.By add 5m M of isocitrate to the media of Aco2-deficient cells,lipid accumulation was efficiently restored morphologically and the m RNA and protein levels of genes related to adipogenesis and TG synthesis were also increased.Meanwhile,mitochondrial biogenesis and ATP content were also increased.These results indicate that supplementation of isocitrate restores adipocyte differentiation in Aco2-deficient cells.To further test whether production of ATP was the limiting factor for the stimulation of adipogenesis by isocitrate,20μm of rotenone was added into isocitrate-treated Aco2-deficient cells,the results show depletion of ATP production abolishes the effect of isocitrate,suggesting that ATP is the rate-limiting factor for isocitrate to restore adipogenesis in Aco2-deficient cells.In summary,our results demonstrate an important capacity for adipocyte Aco2 in adipogenesis,overexpression of Aco2 promotes lipid accumulation in 3T3-L1 cells,deficiency of Aco2 inhibit adipogenesis in adipocytes and supplementation of isocitrate restores adipocyte differentiation in Aco2-deficient cells.On the other hand,mechanistically,the role of Aco2 in adipogenesis is ATP-dependent.By ATP is depleted,the promotive role of Aco2 in adipogenesis is abolished.更多还原