棕榈酸对模拟高原低氧大鼠离体脑线粒体解耦联蛋白活性及质子漏的影响

本文旨在通过观察棕榈酸对模拟高原低氧大鼠离体脑线粒体解耦联蛋白(uncoupling proteins,UCPs)活性的影响及脑线粒体质子漏与膜电位的改变,探讨UCPs在介导游离脂肪酸对低氧时线粒体氧化磷酸化功能改变中的作用.将SpragueDawley大鼠随机分为对照组、急性低氧组和慢性低氧组.低氧大鼠于低压舱内模拟海拔5 000 m高原23 h/d作低氧暴露,分别连续低氧3 d和30 d.用差速密度梯度离心法提取脑线粒体,[3H-GTP法测定UCPs含量与活性,TPMP 电极与Clark氧电极结合法测量线粒体质子漏,罗丹明123荧光法测定线粒体膜电位.结果显示,低氧使脑线粒体内UCPs含量与活性升高、质子漏增加、线粒体膜电位降低;同时,低氧暴露降低脑线粒体对棕榈酸的反应性,UCPs活性的改变率低于对照组,且线粒体UCPs含量、质子漏、膜电位变化率亦出现相同趋势.线粒体质子漏与反映UCPs活性的Kd值呈线性负相关(P<0.01 r=-0.906),与反映UCPs含量的Bmax呈线性正相关(P<0.01,r=0.856),与膜电位呈线性负相关(P<0.01,r=-0.880).以上结果提示,低氧导致的脑线粒体质子漏增加及膜电位降低与线粒体内UCPs活性升高有关,同时低氧暴露能降低脑线粒体对棕榈酸的反应性,提示在高原低氧环境下,游离脂肪酸升高在维持线粒体能量代谢中起着自身保护和调节机制.     

大鼠脑线粒体体外蛋白翻译体系的建立及蛋白产物的鉴定

目的 :建立大鼠脑组织线粒体的体外蛋白合成体系并对其合成产物进行电泳分离和分子量鉴定。方法 :分离大鼠脑组织线粒体 ,用3 H 亮氨酸掺入法探索线粒体体外翻译的最佳条件 ,3 5S 蛋氨酸掺入并对翻译后产物经SDS 聚丙烯酰胺凝胶电泳和放射自显影进行分子量鉴定。结果 :分离的线粒体氧化磷酸化偶联程度高 ,呼吸控制率(RCR)在 3.5～ 5 .5之间 ;体外3 H 亮氨酸的掺入活性在 6 0min内近似线性增长 ,而后维持在一相对稳定水平 ;3 H 亮氨酸的掺入活性随线粒体蛋白浓度而增加 ,而单位线粒体蛋白的掺入活性在 1mg/ml时最高 ;3 5S 蛋氨酸掺入SDS 聚丙烯酰胺凝胶电泳后可观察到清晰的 8条自显影带 ,分子量分别为 (单位Kda) 86、6 6、5 6、43、33、2 9、2 5、18。结论 :用此方法建立的脑线粒体离体翻译反应体系具有高活性和翻译忠实性等特点 ,是研究脑mtDNA在翻译水平的表达及调控的有效方法     

耐力训练抑制急性低氧时骨骼肌线粒体生物能学变化:ROS和UCP3的作用(英文)

骨骼肌线粒体解耦联蛋白3（uncoupling protein3,UCP3）在低氧时的生理作用尚不清楚。本研究观察了大鼠在耐力训练前后,模拟急性高原低氧各时间点的骨骼肌线粒体UCP3 mRNA和蛋白表达、线粒体呼吸功能、活性氧（reactive oxygen species,ROS）产生速率以及锰超氧化物歧化酶（manganese superoxide dismutase,MnSOD）表达和活性的变化。急性低氧导致线粒体一系列生物能学功能障碍。未训练大鼠UCP3蛋白在4h时比静息时升高了60％,而MnSOD蛋白含量及活性在低氧暴露过程中无显著变化;UCP3蛋白上调通过降低电子传递链耦联程度抑制O2-产生,但同时降低了ATP合成效率。耐力训练显著抑制急性低氧诱导的骨骼肌UCP3蛋白上调（67％;S42％）。训练组大鼠的ROS产生速率在低氧2h、4h和6h时显著低于未训练组;MnSOD蛋白含量及活性分别较术训练组提高了50％和34％。训练组人鼠MnSOD上调可增加线粒体对ROS的耐受力,进而抑制UCP3蛋白表达,从而提高氧化磷酸化效率。急性低氧中,未训练组大鼠呼吸控制比（respiratory control ratio,RCR）和磷氧比（ADP to oxygen consumption ratio,P／O）显著降低,而训练组RCR和P／O保持相对稳定。以上结果提示：（1）模拟急性高原低氧可诱导UCP3 mRNA及蛋白表达升高,从而降低升高了的线粒体膜电位（△ψ）,使ROS的产生减少;（2）耐力训练可抑制低氧诱导的UCP3表达上调,提高ROS酶学清除能力,从而提高线粒体氧化磷酸化效率。     

禁食及胰岛素水平对大鼠解偶联蛋白-1、2、3基因表达的影响

 为探讨禁食和胰岛素对解偶联蛋白 - 1、2、3基因 (UCP1 ,2 ,3)表达的影响 ,应用 RT- PCR方法观察了在不同禁食时间和应用胰岛素条件下大鼠白色脂肪组织、棕色脂肪组织和骨骼肌中 UCP1 ,2 ,3m RNA水平的变化 .UCP1基因只在大鼠棕色脂肪组织中表达 .UCP2 ,3基因在三种组织中均有表达 ,在白色脂肪组织中以 UCP2表达为主 ;在骨骼肌中以 UCP3表达为主 .过夜禁食使棕色脂肪组织 UCP1 ,3m RNA水平明显下降 (P<0 .0 1 ) ;UCP2 m RNA水平在三种组织中均呈上升反应 ,以白色脂肪组织中表现最为明显 (P<0 .0 5) ;而对白色脂肪组织和骨骼肌中 UCP3基因表达无明显影响 .禁食时间延长至 48h,除棕色脂肪组织中 UCP2 ,3基因有明显下降外 ,各组织中UCPs基因表达基本调节至正常或高于对照组水平 .胰岛素对 UCPs基因表达水平有一定的上调作用 ,这一作用对棕色脂肪组织 UCPs各基因及骨骼肌中 UCP3基因表现得尤为明显 (P<0 .0 5) .大鼠 UCPs基因表达有一定的组织特异性 ;禁食时间对三种组织中 UCPs各成员基因表达的影响有时相上的区别 ;胰岛素可以调 UCPs各成员基因的表达 .结果反映了 UCPs各成员在能量代谢调节上的不同作用 ,这为理解膳食 -产热与体重调节的关系 ,及其能量代谢平衡与疾病关系提供了实验依据     

鲢鱼解偶联蛋白2全长cDNA序列的克隆及其组织表达

从淡水食毒藻鱼类鲢鱼(Hypophthalmichthysmolitrix)肝脏,通过简并引物克隆解偶联蛋白2(un-couplingprotein2,UCP2)cDNA核心序列,应用5′RACE和3′RACE技术分别扩增该序列的5′末端和3′末端序列,最后通过序列拼接获得鲢鱼肝脏UCP2cDNA全序列。序列分析结果表明,鲢鱼肝脏UCP2cDNA全长1452bp,其中5′-UTR长337bp,3′-UTR长182bp,编码区933bp,编码310个氨基酸,推测的氨基酸序列包含线粒体内膜载体蛋白3个特征结构及解偶联蛋白(UCPs)的特征序列。对鲢鱼不同组织UCP2的表达调控研究发现,鲢鱼组织UCP2基因在肠道、肝脏、肌肉、脂肪组织均大量表达,而在脑组织表达量较低,这与鲢鱼体内微囊藻毒素在这几个组织的分布完全一致,表明UCP2的功能可能与抑制微囊藻毒素引发过量活性氧(ROS)生成有关。     

何首乌提取物减轻大鼠脑缺血再灌注损伤和上调脑内UCP4蛋白水平

目的研究何首乌提取物对脑缺血再灌注损伤UCP4的影响,初步探讨其可能的作用机制。方法健康雄性SD大鼠采用线栓法复制局灶性脑缺血(MCAO)再灌注损伤模型,缺血2h后再灌注6h或24h,部分缺血再灌注模型大鼠分别灌胃不同浓度何首乌口服液。免疫组织化学染色和Western blot检测脑内UCP4表达,TTC染色检测脑梗死面积。结果在脑缺血再灌注6h后损伤,UCP4蛋白在海马内表达增高,再灌注24h后表达降低;何首乌提取物能浓度依赖性减少脑缺血再灌注损伤的脑梗死面积和上调UCP4表达。结论何首乌提取物能明显改善大鼠脑缺血再灌注损伤,其机制可能与脑内线粒体蛋白UCP4表达升高,保护神经元有关。     

解偶联蛋白4的线粒体保护作用

线粒体解偶联蛋白(UCPs)是近年来发现的线粒体膜蛋白家族中的新成员.研究表明,解偶联蛋白4(UCP4)有调节线粒体膜电位、减少氧自由基的生成、调节细胞内钙离子浓度等作用,受细胞代谢、甲状腺激素,以及儿茶酚胺等调节.UCP4主要分布于大脑皮质和海马区,可能在脑血管病、精神分裂症、变性病等线粒体易受损的疾病中起重要作用.     

猪脑突触质膜糖皮质激素膜结合位点的测定

利用放射配体结合法,测定了猪脑六个不同脑区突触质膜糖皮质激素膜结合位点(GCM-BS)的最大结合量(Bmax)和平衡解离常数(Kd)。结果表明:猪的大脑、小脑、中脑、丘脑、下丘脑和尾状核的突触质膜GCMBS的Kd都在200nmol/L左右,提示它们对[~3H]皮质酮的亲和力基本相同;而各脑区内GCMBS的Bmax相差较大,从218.98fmol/mg蛋白至486.95fmol/mg蛋白不等,说明不同脑区内所含有的GCMBS的数量不同。     

鲢鱼解偶联蛋白2全长cDNA序列的克隆及其组织表达

从淡水食毒藻鱼类鲢鱼(Hypophthalmichthys molitrix)肝脏，通过简并引物克隆解偶联蛋白2(uncoupling protein 2，UCP2) cDNA核心序列，应用5′RACE和3′RACE技术分别扩增该序列的5′末端和3′末端序列，最后通过序列拼接获得鲢鱼肝脏UCP2 cDNA全序列。序列分析结果表明，鲢鱼肝脏UCP2 cDNA全长1 452 bp，其中5′-UTR长337 bp，3′-UTR长182 bp，编码区933 bp，编码310个氨基酸，推测的氨基酸序列包含线粒体内膜载体蛋白3个特征结构及解偶联蛋白(UCPs)的特征序列。对鲢鱼不同组织UCP2的表达调控研究发现，鲢鱼组织UCP2基因在肠道、肝脏、肌肉、脂肪组织均大量表达，而在脑组织表达量较低，这与鲢鱼体内微囊藻毒素在这几个组织的分布完全一致，表明UCP2的功能可能与抑制微囊藻毒素引发过量活性氧(ROS)生成有关。     

乌灵胶囊对抑郁大鼠脑组织中乙酰化H3 及5-HTT、TH 表达的影响

目的:观察乌灵胶囊对抑郁大鼠脑组织中乙酰化组蛋白H3及5-羟色胺受体(5-HTT)、酪氨酸羟化酶(tyrosine hydroxylase,TH)表达的影响。方法:SD大鼠随机分为对照组、模型组及乌灵胶囊低、高剂量组。取大鼠脑组织,分别采用实时荧光定量PCR法和western blot法检测脑组织中乙酰化组蛋白H3、组蛋白H3,5-HTT、TH蛋白和mRNA的表达。结果:乌灵胶囊明显增强大鼠脑组织中乙酰化组蛋白比例和5-HTT、TH蛋白和mRNA的表达(P<0.05)。结论:乌灵胶囊治疗抑郁症的机制可能与升高脑组织乙酰化组蛋白含量,从而促进5-HTT、TH的表达有关。     

A comparative study of the inhibitory effects of purine nucleotides and carboxyatractylate on the uncoupling protein-3 and adenine nucleotide translocase

Uncoupling proteins (UCPs) mediate fatty acid-induced proton cycling in mitochondria, which is stimulated by superoxide and inhibited by GDP. Fatty acid anions can also be transported by adenine nucleotide translocase (ANT), thus resulting in the uncoupling of oxidative phosphorylation. In the present work, an attempt was made to distinguish between the protonophoric activity of UCP3 and that of ANT using inhibition analysis. This study was carried out using mitochondria from skeletal muscles of hibernating Yakut ground squirrel, which have a significant level of UCP3 mRNA. We found that millimolar concentrations of GDP, which is considered to be a specific inhibitor of UCPs, slightly recoupled the mitochondrial respiration and restored the membrane potential. Addition of the specific ANT inhibitor CAT (carboxyatractylate), in micromolar concentration, prior to GDP prevented its recoupling effect. Moreover, GDP and ADP exhibited a competitive kinetic behavior with respect to ANT. In brown adipose tissue, CAT did not prevent the UCP1-iduced increase in chloride permeability and the inhibitory effect of GDP, thus confirming the inability of CAT to affect UCP1. These results allow us to conclude that the recoupling effect of purine nucleotides on skeletal muscle mitochondria of hibernating ground squirrels can be explained by interaction of the nucleotides with ANT, whereas UCP3 is not involved in the process.     

UCP2 and UCP3 rise in starved rat skeletal muscle but mitochondrial proton conductance is unchanged

The relationship between UCP2 and UCP3 expression and mitochondrial proton conductance of rat skeletal muscle was examined. Rats were starved for 24 h and the levels of UCP2 and UCP3 mRNA and UCP3 protein were determined by Northern and Western blots. Proton conductance was measured by titrating mitochondrial respiration rate and membrane potential with malonate. Starvation increased UCP2 and UCP3 mRNA levels more than 5-fold and 4-fold, respectively, and UCP3 protein levels by 2-fold. However, proton conductance remained unchanged. These results suggest either that Northern and Western blots do not reflect the levels of active protein or that these UCPs do not catalyse the basal proton conductance in skeletal muscle mitochondria.     

Functional characterization of UCP1 in mammalian HEK293 cells excludes mitochondrial uncoupling artefacts and reveals no contribution to basal proton leak

Mechanistic studies on uncoupling proteins (UCPs) not only are important to identify their cellular function but also are pivotal to identify potential drug targets to manipulate mitochondrial energy transduction. So far, functional and comparative studies of uncoupling proteins in their native environment are hampered by different mitochondrial, cellular and genetic backgrounds. Artificial systems such as yeast ectopically expressing UCPs or liposomes with reconstituted UCPs were employed to address crucial mechanistic questions but these systems also produced inconsistencies with results from native mitochondria. We here introduce a novel mammalian cell culture system (Human Embryonic Kidney 293 - HEK293) to study UCP1 function. Stably transfected HEK293 cell lines were derived that contain mouse UCP1 at concentrations comparable to tissue mitochondria. In this cell-based test system UCP1 displays native functional behaviour as it can be activated with fatty acids (palmitate) and inhibited with purine nucleotides guanosine-diphosphate (GDP). The catalytic centre activity of the UCP1 homodimer in HEK293 is comparable to activities in brown adipose tissue supporting functionality of UCP1. Importantly, at higher protein levels than in yeast mitochondria, UCP1 in HEK293 cell mitochondria is fully inhibitable and does not contribute to basal proton conductance, thereby emphasizing the requirement of UCP1 activation for therapeutic purposes. These findings and resulting analysis on UCP1 characteristics demonstrate that the mammalian HEK293 cell system is suitable for mechanistic and comparative functional studies on UCPs and provides a non-confounding mitochondrial, cellular and genetic background.     

Functional characterization of the mitochondrial uncoupling proteins from the white shrimp Litopenaeus vannamei

Mitochondrial uncoupling proteins (UCPs) play an essential role in dissipating the proton gradient and controlling the mitochondrial inner membrane potential. When active, UCPs promote proton leak across the inner membrane, oxidative phosphorylation uncoupling, oxygen uptake increase and decrease the ATP synthesis. Invertebrates possess only isoforms UCP4 and UCP5, however, the role of these proteins is not clear in most species since it may depend on the physiological needs of each animal. This study presents the first functional characterization of crustacean uncoupling proteins from the white shrimp Litopenaeus vannamei LvUCP4 and LvUCP5. Free radicals production in various shrimp organs/tissues was first evaluated, and mitochondria were isolated from shrimp pleopods. The oxygen consumption rate, membrane potential and proton transport of the isolated non-phosphorylating mitochondria were used to determine LvUCPs activation/inhibition. Results indicate that UCPs activity is stimulated in the presence of 4-hydroxyl-2-nonenal (HNE) and myristic acid, and inhibited by the purine nucleotide GDP. A hypoxia/re-oxygenation assay was conducted to determine whether UCPs participate in shrimp mitochondria response to oxidative stress. Isolated mitochondria from shrimp at re-oxygenation produced large quantities of hydrogen peroxide and higher levels of both LvUCPs were immunodetected. Results suggest that, besides the active response of the shrimp antioxidant system, UCP-like activity is activated after hypoxia exposure and during re-oxygenation. LvUCPs may represent a mild uncoupling mechanism, which may be activated before the antioxidant system of cells, to early control reactive oxygen species production and oxidative damage in shrimp.     

Comparative analysis of uncoupling protein 4 distribution in various tissues under physiological conditions and during development

UCP4 is a member of the mitochondrial uncoupling protein subfamily and one of the three UCPs (UCP2, UCP4, UCP5), associated with the nervous system. Its putative functions include thermogenesis, attenuation of reactive oxidative species (ROS), regulation of mitochondrial calcium concentration and involvement in cell differentiation and apoptosis. Here we investigate UCP4's subcellular, cellular and tissue distribution, using an antibody designed specially for this study, and discuss the findings in terms of the protein's possible functions. Western blot and immunohistochemistry data confirmed that UCP4 is expressed predominantly in the central nervous system (CNS), as previously shown at mRNA level. No protein was found in heart, spleen, stomach, intestine, lung, thymus, muscles, adrenal gland, testis and liver. The reports revealing UCP4 mRNA in kidney and white adipose tissue were not confirmed at protein level. The amount of UCP4 varies in the mitochondria of different brain regions, with the highest protein content found in cortex. We show that UCP4 is present in fetal murine brain tissue as early as embryonic days 12-14 (E12-E14), which coincides with the beginning of neuronal differentiation. The UCP4 content in mitochondria decreases as the age of mice increases. UCP4 preferential expression in neurons and its developmental expression pattern under physiological conditions may indicate a specific protein function, e.g. in neuronal cell differentiation.     

Functional analysis of skunk cabbage SfUCPB, a unique uncoupling protein lacking the fifth transmembrane domain, in yeast cells

Skunk cabbage, Symplocarpus foetidus, expresses two uncoupling proteins (UCPs), termed SfUCPA and SfUCPB, in the thermogenic organ spadix. SfUCPB exhibits unique structural features characterized by the absence of the putative fifth transmembrane domain (TM5) observed in SfUCPA, which is structurally similar to UCP1, and is abundantly expressed in the thermogenic spadix. Here, we conducted a series of comparative analyses of UCPs with six transmembrane domains, SfUCPA and rat UCP1, and TM5-deficient SfUCPB, using a heterologous yeast expression system. All UCPs were successfully expressed and targeted to the mitochondria, although the expression level of SfUCPB protein was approximately 10% of rat UCP1. The growth rate, mitochondrial membrane potential, and ATP content were significantly lower in cells expressing SfUCPB than in those expressing rat UCP1 and SfUCPA. These results suggest that SfUCPB, a novel TM5-deficient UCP, acts as an uncoupling protein in yeast cells.     

Evidence for Involvement of Uncoupling Proteins in Cerebral Mitochondrial Oxidative Phosphorylation Deficiency of Rats Exposed to 5,000 m High Altitude

The present study aimed to investigate the change of proton leak and discuss the role of cerebral uncoupling proteins (UCPs) and its regulatory molecules non-esterified fatty acid (NEFA) in high altitude mitochondrial oxidative phosphorylation deficiency. The model group animals were exposed to acute high altitude hypoxia, and the mitochondrial respiration, protein leak, UCPs abundance/activity and cerebral NEFA concentration were measured. We found that in the model group, cerebral mitochondrial oxidative phosphorylation was severely impaired with decreased ST3 respiration rate and ATP pool. Proton leak kinetics curves demonstrated an increase in proton leak; GTP binding assay pointed out that total cerebral UCPs activity significantly increased; Q-PCR and western blot showed upregulated expression of UCP4 and UCP5. Moreover, cerebral NEFA concentration increased. In conclusion, UCPs mediated proton leak is closely related to cerebral mitochondria oxidative phosphorylation deficiency during acute high altitude hypoxia and NEFA is involved in this signaling pathway.     

A signalling role for 4-hydroxy-2-nonenal in regulation of mitochondrial uncoupling

Oxidative stress and mitochondrial dysfunction are associated with disease and aging. Oxidative stress results from overproduction of reactive oxygen species (ROS), often leading to peroxidation of membrane phospholipids and production of reactive aldehydes, particularly 4-hydroxy-2-nonenal. Mild uncoupling of oxidative phosphorylation protects by decreasing mitochondrial ROS production. We find that hydroxynonenal and structurally related compounds (such as trans-retinoic acid, trans-retinal and other 2-alkenals) specifically induce uncoupling of mitochondria through the uncoupling proteins UCP1, UCP2 and UCP3 and the adenine nucleotide translocase (ANT). Hydroxynonenal-induced uncoupling was inhibited by potent inhibitors of ANT (carboxyatractylate and bongkrekate) and UCP (GDP). The GDP-sensitive proton conductance induced by hydroxynonenal correlated with tissue expression of UCPs, appeared in yeast mitochondria expressing UCP1 and was absent in skeletal muscle mitochondria from UCP3 knockout mice. The carboxyatractylate-sensitive hydroxynonenal stimulation correlated with ANT content in mitochondria from Drosophila melanogaster expressing different amounts of ANT. Our findings indicate that hydroxynonenal is not merely toxic, but may be a biological signal to induce uncoupling through UCPs and ANT and thus decrease mitochondrial ROS production.