UCPs在高原缺氧大鼠脑线粒体能量代谢中的作用

发布时间：2018-08-12 16:36

【摘要】： 线粒体氧化磷酸化是由呼吸链在将电子传递给氧的过程中将质子从内膜基质转到内膜外,形成跨膜质子电动势(△P),驱动ATP合成酶催化ADP和无机磷合成ATP。质子还可通过内膜上的另一质子通道——脱偶联蛋白(Uncoupling Proteins, UCPS)漏回到基质中,形成质子漏,降低△P,使氧化磷酸化脱偶联,减少ATP的生成,从而降低用氧效率,这部分氧耗为“无效氧耗”。高原缺氧时线粒体脱偶联增强,膜电位降低,能量生成减少,导致机体功能和代谢障碍。脱偶联蛋白4(Uncoupling Proteins 4,UCP4)和脱偶联蛋白5(Uncoupling Proteins 5,UCP5)是特异存在于哺乳动物脑组织中的UCPs家族成员,占脑中UCPs的84%以上。游离脂肪酸通过与UCPs分子构象中某些位点结合而促进棕色脂肪内UCP1,骨骼肌中UCP2和UCP3的活性,是潜在的UCP4、UCP5的激动剂。本实验通过棕榈酸脑组织块体外干预模型和棕榈酸游离脑线粒体干预模型,观察外源性游离脂肪酸对UCPs的活性和“含量”的影响及其在高原缺氧大鼠脑线粒体能量合成改变的作用,以探讨UCPs在缺氧大鼠脑线粒体能量生成和氧利用效率中的作用及调控机制。方法分别建立棕榈酸脑组织块体外干预模型和棕榈酸脑游离线粒体干预模型,在观察了棕榈酸对大鼠脑组织UCP4和UCP5 mRNA和蛋白表达的时效性影响以及对游离脑线粒体氧化磷酸化影响的时效性和量效性基础上,进一步探讨了棕榈酸对模拟高原缺氧大鼠脑UCPs表达、活性及其与线粒体呼吸氧耗和能量生成的关系。健康雄性SD大鼠暴露于模拟海拔5000米高原低压舱内,23h/d,分别连续缺氧3天(急性组)和30天(慢性组),同时设立对照组。心脏取血后,分别在平原和模拟高原低压舱断头处死,取大脑半球,分别切成1～2×2～3mm2的组织碎块和直接分离线粒体,分别以100μmol/L的棕榈酸进行脑组织块体外干预和游离脑线粒体体外干预,以Clark氧电极法测定线粒体氧化呼吸活性,TPMP+电极与Clark氧电极结合测质子漏,寡霉素抑制法测定F0F1-ATP酶活性,罗丹明123法测定线粒体膜电位,高压液相色谱法分析脑组织线粒体内腺苷酸含量,[3H]-GTP结合法测定脑组织UCPs的活性。RT-PCR和Westernblot分别测定脑组织干预后UCP4、UCP5 mRNA和蛋白表达。同时,以铜离子法测定缺氧大鼠血清、脑组织匀浆液和线粒体内游离脂肪酸含量。结果 1.100μmol/L的棕榈酸大鼠脑组织块体外干预时间达30min时,UCP4和UCP5 mRNA表达达其峰值,而蛋白表达亦显著升高,线粒体氧化磷酸化呼吸效率显著降低。棕榈酸大鼠游离脑线粒体体外干预,当浓度在0.1 mmol/L内,时间在1min内时与线粒体氧化呼吸存在量效关系。 2.缺氧能增强大鼠脑组织UCPs活性,急性缺氧组Kd值降低41.24%,而Bmax值升高1.56倍。棕榈酸干预能进一步增加各组UCPs的活性,但使急性组增加幅度最低,Kd值仅下降13.96%,而Bmax则仅升高16.01%。 3.缺氧使大鼠血清、脑组织匀浆、线粒体内游离脂肪酸含量升高,以急性组升高最明显,分别达到51.36%, 243.35%和69.49%,慢性组较急性组有所下降,但仍高于对照组。相关性分析显示,大鼠血清、脑组织、线粒体游离脂肪酸含量与反映UCPs活性的Kd值呈线性负相关,与反映UCPs“含量”的Bmax呈线性正相关(脑组织游离脂肪酸含量与UCPs活性的相关系数最高)。 4.缺氧组脑线粒体ST3、RCR、OPR、P/O和MMP明显降低,ST4、质子漏则明显升高。棕榈酸可进一步增加各组呼吸氧耗和质子漏,降低MMP,其中对急性缺氧组的影响最小,ST3和ST4分别升高5.12%和38.69%,RCR、OPR和P/O分别升高24.64%、3.19%和3.96%,MMP仅降低6.91%。 5.缺氧组脑线粒体F0F1-ATP酶活性、ATP含量、ATP/ADP和ATP/总腺苷酸比值显著降低,以急性组降低最显著,分别降低43.32%、27.79%、16.39%和28.67%。棕榈酸可使急性组总腺苷酸池(ATP+ADP+AMP)、(ATP+ADP)池含量分别降低30.90%和49.29%以及各组ATP/ADP均升高,且急性组升高163.04%;同时各组ATP/总腺苷酸池比值均降低,对照组、急性组能荷均分别降低36.91%和13.64%。 6.缺氧组脑UCP4、UCP5mRNA和蛋白表达增高,其中急性组上调幅度最大,UCP4mRNA和蛋白表达分别为对照组的19.04倍和16.95倍,UCP5 mRNA以及蛋白则分别为对照组的2.17倍和4.71倍。棕榈酸体外干预进一步增强各组UCP4、UCP5mRNA和蛋白表达,但急性组升高幅度最小。结论 1.棕榈酸可直接影响脑线粒体UCPs活性和UCP4和UCP5 mRNA和蛋白表达,增强质子漏和脱偶联呼吸,进而影响脑线粒体的氧化磷酸化功能,降低氧化磷酸化效率,且有时间和剂量依赖性。缺氧暴露在一定程度上可削弱棕榈酸促UCPs活性和含量的效能。 2.缺氧可增加脑线粒体UCPs活性,以及UCP4、UCP5 mRNA和蛋白表达,与缺氧时血及脑内游离脂肪酸代谢改变有关。缺氧时游离脂肪酸升高——UCPs活性增强——解耦联——ATP生成下降为缺氧时脑能量合成障碍的中心环节。全文总结模拟高原缺氧暴露可使大鼠血清、脑组织及线粒体内游离脂肪酸含量升高,脑线粒体UCPs活性和含量增加,脑UCP4、UCP5 mRNA和蛋白表达增强,质子漏增强,膜电位降低,从而使“无效氧耗”增加,氧化磷酸化效率降低,线粒体能量合成减少;棕榈酸则可进一步升高缺氧大鼠脑线粒体UCPs活性与含量,增强脑UCP4、UCP5 mRNA和蛋白表达,从而增加质子漏,降低膜电位,增强脱偶联,具有降低氧化磷酸化效率和能量生成效率的作用。棕榈酸的这些作用与缺氧暴露有关,缺氧暴露可弱化棕榈酸的作用。实验揭示了模拟高原缺氧时游离脂肪酸——UCPs表达、含量及活性——线粒体呼吸氧耗——ATP生成之间的相互关系,提示游离脂肪酸——UCPs相互作用是缺氧时组织能量代谢障碍的重要环节之一。
[Abstract]:Mitochondrial oxidative phosphorylation is the transfer of protons from the endometrial matrix to the outer membrane by the respiratory chain during electron transfer to oxygen, forming a transmembrane proton electromotive force (delta P), which drives ATP synthase to catalyze the synthesis of ATP from ADP and inorganic phosphorus. Back in the matrix, proton leaks are formed, Delta P is reduced, oxidative phosphorylation is decoupled, ATP is reduced, and oxygen use efficiency is reduced. This part of oxygen consumption is "ineffective oxygen consumption". UCP4 and UCP5 are members of the UCPs family specifically present in mammalian brain tissues, accounting for more than 84% of the UCPs in the brain. Free fatty acids promote the activity of UCP1 in brown fat, UCP2 and UCP3 in skeletal muscle by binding to certain sites in the conformation of UCPs. They are potential agonists of UCP4 and UCP5. In order to investigate the effect of exogenous free fatty acids on the activity and content of UCPs and the change of mitochondrial energy synthesis in the brain of hypoxic rats at high altitude, the in vitro intervention model of palmitic acid brain block and the model of palmitic acid free brain mitochondrial intervention were used to observe the effect of exogenous free fatty acids on the activity and content of UCPs. The role and regulation mechanism of efficiency.
Method
The in vitro intervention model of palmitic acid brain tissue block and the model of palmitic acid brain free mitochondria intervention were established. The effect of palmitic acid on the expression of UCP4 and UCP5 mRNA and protein in rat brain tissue and the effect of palmitic acid on the oxidative phosphorylation of free brain mitochondria were observed. Healthy male SD rats were exposed to simulated altitude hypobaric chamber at 5000 m for 23 h/d for 3 days (acute group) and 30 days (chronic group) respectively, and control group was set up. After blood collection, the rats were exposed to simulated altitude hypobaric chamber for 3 days (acute group) and 30 days (chronic group). Cerebral hemisphere was cut into 1-2 *2-3 mm2 tissue fragments and mitochondria were separated directly. Brain tissue fragments and free brain mitochondria were interfered with palmitic acid at 100 micromol/L in vitro. Mitochondrial oxidative respiratory activity was measured by Clark oxygen electrode, proton leakage was detected by TPMP + electrode and Clark oxygen electrode. The activity of F0F1-ATPase was measured by inhibiting factor assay, mitochondrial membrane potential by rhodamine 123, adenylate content in brain mitochondria by high performance liquid chromatography, and UCPs activity by combining [3H]-GTP. The expression of UCP4 and UCP5 mRNA and protein were measured by RT-PCR and Western blot respectively. The contents of free fatty acids in serum, brain homogenate and mitochondria were measured in rats.
Result
The expression of UCP4 and UCP5 mRNA reached its peak value, and the expression of UCP4 and UCP5 protein increased significantly, and the respiratory efficiency of mitochondria oxidative phosphorylation decreased significantly. The free cerebral mitochondria of palmitic acid Rats Intervened in vitro when the concentration of palmitic acid was within 0.1 mmol/L and the time was within 1 minute. There is a dose effect relationship.
2. Hypoxia could enhance the activity of UCPs in rat brain tissue. The Kd value decreased by 41.24% in acute hypoxia group, while the Bmax value increased by 1.56 times. Palmitic acid intervention could further increase the activity of UCPs in each group, but the increase was the lowest in acute group. The Kd value decreased only by 13.96%, while the Bmax value increased only by 16.01%.
3. Hypoxia increased the content of free fatty acids in serum, brain homogenate and mitochondria of rats, the highest in acute group was 51.36%, 243.35% and 69.49% respectively. The content of free fatty acids in serum, brain tissue and mitochondria of chronic group was lower than that of acute group, but still higher than that of control group. There was a linear negative correlation between D value and Bmax reflecting UCPs content (the highest correlation coefficient between free fatty acid content and UCPs activity in brain tissue).
4. Mitochondrial ST3, RCR, OPR, P/O and MMP decreased significantly in hypoxic group, while ST4 and proton leakage increased significantly. Palmitic acid could further increase respiratory oxygen consumption and proton leakage, and reduce MMP. Among them, ST3 and ST4 increased by 5.12% and 38.69%, RCR, OPR and P/O increased by 24.64%, 3.19% and 3.96% respectively, while MMP decreased by only 6.91%.
5. The activity of F0F1-ATPase, ATP content, ATP/ADP and ATP/total adenylate ratio in the hypoxic group were significantly decreased, with the most significant decrease in the acute group, 43.32%, 27.79%, 16.39% and 28.67% respectively. Palmitic acid could decrease the total adenylate pool (ATP+ADP+AMP), ATP+ADP pool (ATP+ADP) content by 30.90% and ATP/ADP ratio by 49.29% in the acute group. At the same time, ATP / total adenylate pool ratio of each group decreased, while energy charge of control group and acute group decreased by 36.91% and 13.64% respectively.
6. The expression of UCP4, UCP5 mRNA and protein in the brain of hypoxic group increased significantly. The expression of UCP4 mRNA and protein was 19.04 and 16.95 times higher in the acute group than in the control group. The expression of UCP5 mRNA and protein was 2.17 and 4.71 times higher in the hypoxic group than that in the control group. The minimum amplitude.
conclusion
1. Palmitic acid can directly affect the activity of UCPs and the expression of UCP4 and UCP5 mRNA and protein in brain mitochondria, enhance proton leakage and decoupling respiration, then affect the oxidative phosphorylation function of brain mitochondria, reduce the efficiency of oxidative phosphorylation, and it is time and dose dependent. Hypoxia exposure can weaken the activity and content of palmitic acid-induced UCPs to a certain extent. Efficiency.
2. Hypoxia can increase the activity of UCPs in brain mitochondria, and the expression of UCP4, UCP5 mRNA and protein, which is related to the changes of free fatty acid metabolism in blood and brain during hypoxia.
A summary of the full text
Simulated high altitude hypoxia exposure can increase the free fatty acid content in serum, brain tissue and mitochondria, increase the activity and content of UCPs in brain mitochondria, increase the expression of UCP4 and UCP5 mRNA and protein, increase the proton leakage and decrease the membrane potential, so that the "ineffective oxygen consumption" increases, the oxidative phosphorylation efficiency decreases, and mitochondrial energy synthesis decreases. Palmitic acid can further increase the activity and content of UCPs in the brain mitochondria of hypoxic rats, enhance the expression of UCP4, UCP5 mRNA and protein, thereby increasing proton leakage, reducing membrane potential, enhancing decoupling, and reducing the efficiency of oxidative phosphorylation and energy production. The experiment reveals the relationship between the expression, content and activity of free fatty acid-UCPs, oxygen consumption of mitochondria and ATP production during simulated altitude hypoxia, suggesting that the interaction of free fatty acid-UCPs is one of the important links of energy metabolism disorder during hypoxia.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R363

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