解偶联蛋白2对缺血性脑卒中脑保护作用的研究进展

解偶联蛋白2(UCP2)是线粒体内膜上的一种载体蛋白,在脑内广泛分布.近年来的研究显示,UCP2可通过调控氧化应激反应、线粒体功能、细胞凋亡、物质能量与代谢等参与缺血性脑卒中的病理过程.本文就UCP2对缺血性脑卒中的脑保护机制进行归纳.     

解偶联蛋白在能量代谢和体温调节中的作用研究进展

棕色脂肪组织（BAT）是哺乳动物体内非战栗产热的主要来源，对于维持动物的体温和能量平衡起重要作用。位于BAT线粒体内膜的解偶联蛋白（UCP）是决定BAT功能的关键因素，其作为质子通道驱散氧化呼吸时形成的H^ 梯度，从而增加呼吸，阻止ATP形成，通过将呼吸链与ATP产生过程解偶联来影响体内能量平衡。这些解偶联蛋白的生物学特性是近年来研究的热点，迄今为止所发现的解偶联蛋白家族成员，主要有UCP1、UCP2、UCP3、UCP4以及UCP5（脑线粒体转运蛋白，BM-CP1）。对年来年关于UCP的动物学研究以及在人体中的研究两个领域中的实验结论做了一个总结，对UCP在能量代谢和体温调节中的作用进行了系统的探讨和展望。     

解偶联蛋白在能量代谢和体温调节中的作用研究进展

棕色脂肪组织(BAT)是哺乳动物体内非战栗产热的主要来源,对于维持动物的体温和能量平衡起重要作用。位于BAT线粒体内膜的解偶联蛋白(UCP)是决定BAT功能的关键因素,其作为质子通道驱散氧化呼吸时形成的H+梯度,从而增加呼吸,阻止ATP形成,通过将呼吸链与ATP产生过程解偶联来影响体内能量平衡。这些解偶联蛋白的生物学特性是近年来研究的热点,迄今为止所发现的解偶联蛋白家族成员,主要有UCP1、UCP2、UCP3、UCP4以及UCP5(脑线粒体转运蛋白,BM-CP1)。对近年来关于UCP的动物学研究以及在人体中的研究两个领域中的实验结论做了一个总结,对UCP在能量代谢和体温调节中的作用进行了系统的探讨和展望。     

低氧复合运动对大鼠骨骼肌解偶联蛋白3表达及线粒体功能的影响

目的:观察单纯低氧及低氧复合运动对大鼠骨骼肌线粒体解偶联蛋白3(UCP3)表达的影响,并探讨其对线粒体能量转换和活性氧(ROS)生成的影响。方法:30只健康雄性SD大鼠随机分为:常氧对照组(NC,n=10)、单纯低氧组(HC,n=10)和低氧复合运动训练组(HT,n=10)。低氧干预为常压低氧帐篷,模拟11.3%的氧浓度,运动干预为低氧帐篷内53%VO2max强度的跑台训练,1h/d。4周后测定线粒体呼吸功能、ATP合成酶活力、ROS生成速率、UCP3mRNA和UCP3蛋白表达。结果:HC与NC组比较,态3呼吸速率(ST3)、呼吸控制比(RCR)、磷氧比(ADP/O)、ATP合成酶活力、UCP3mRNA和蛋白表达均显著降低(P<0.05—0.01),ROS生成速率升高(P<0.05)。HT与HC组比较,ST3、RCR、ADP/O、ATP合成酶活力、UCP3mRNA和蛋白表达均显著升高(P<0.05—0.01),ROS生成速率降低(P<0.05)。结论:低氧复合运动可上调骨骼肌线粒体UCP3表达,抑制ROS过度生成,并通过上调ATP合成酶活力,保持线粒体能量转换效率。     

基于线粒体PCR基因芯片的骨关节炎发病机制

背景:软骨细胞具有修复受损软骨组织和维持软骨完整性的重要作用。线粒体功能障碍与细胞凋亡、老化和骨关节炎的病理过程密切相关。目的:通过线粒体PCR芯片技术研究骨性关节炎软骨细胞中线粒体基因的差异表达。方法:收集骨性关节炎患者及正常成人车祸后截肢者的关节软骨细胞,经过细胞的提取和培养、RNA提取和质量检测、mRNA提取和cDNA合成等处理后进行实时定量PCR检测。结果与结论:所检测到的84个与线粒体相关的待检测基因中有18个线粒体基因在骨性关节炎中发生了显著改变。以骨性关节炎患者软骨细胞线粒体基因相对正常成人软骨细胞线粒体基因的表达倍数表示基因表达的改变情况,其中倍数改变和倍数调节均大于2的上调基因有BBC3,BCL2,SLC25A37等15个,倍数改变小于0.5和倍数调节小于-2的下调基因有CPT1B,SLC25A16,SLC25A24共3个。18个线粒体差异基因功能分类如下:膜极化和电位:BCL2,BCL2L1,TP53,UCP1,UCP3;线粒体转运功能:BCL2,BCL2L1,CPT1B,FXC1(TIMM10B),MFN2,STARD3,TP53,UCP1,UCP3;小分子转运功能:SLC25A16,SLC25A2,SLC25A24,SLC25A31,SLC25A37;靶蛋白:FXC1(TIMM10B),MFN2;线粒体蛋白转入:COX18,FXC1(TIMM10B);内膜转运:FXC1(TIMM10B),TIMM17B;线粒体裂变和融合:COX18,MFN2;线粒体局限化:MFN2;凋亡基因:BBC3,BCL2,BCL2L1,SOD2,P53。结果表明,骨性关节炎软骨细胞中线粒体发生了明显的能量代谢功能障碍。     

解偶联蛋白3在2型糖尿病发生发展中的作用

解偶联蛋白3(UCP3)是解偶联蛋白家族成员之一,系线粒体内膜上阴离子转运蛋白,主要通过质子漏的作用降低线粒体膜内外电化学梯度,影响电子呼吸链,从而使ATP和活性氧的产生减少,使能量以热能的形式释放。UCP3主要在骨骼肌中表达,而骨骼肌是机体外周摄取葡萄糖的主要组织,同时骨骼肌胰岛素抵抗是2型糖尿病患者的主要缺陷,故人们推测,解偶联蛋白3可能在2型糖尿病的发生发展中发挥重要作用。深入探索UCP3在2型糖尿病中的作用有助于为2型糖尿病的治疗提供一个新的治疗靶点。     

甲状腺激素减少对大鼠肾脏解耦联蛋白-2表达的影响

目的 观察甲状腺激素减少时肾脏线粒体解耦联蛋白-2(UCP2)表达的改变,探讨甲状腺激素减少时肾脏损伤的发生机制. 方法 按随机数字表法将Wistar大鼠分为对照组和甲状腺激素减少组(甲减组),每组10只.采用低碘饮食复制甲状腺功能减退大鼠模型,对照组摄碘量10.00 μg/d,甲减组1.24 μg/d,两组均适应喂养1周,条件饲养3个月后取血测定甲状腺功能指标,取肾脏组织,用免疫组化法及逆转录-聚合酶链反应(RT-PCR)测定UCP2的蛋白及mRNA表达. 结果 与对照组比较,甲减组促甲状腺激素(TSH,mU/L)明显上升(4.88±1.37比1.65±0.33,P<0.05),三碘甲状腺原氨酸总量(TT3)、甲状腺素总量(TT4)、游离三碘甲状腺原氨酸(FT3)、游离甲状腺素(FT4)均明显下降[TT3(nmol/L):0.54±0.07比0.98±0.17;TT4(nmol/L):7.82±2.18比48.78±3.65;FT3(pmol/L):2.28±0.22比2.99±0.10;FT4(pmol/L):11.38±1.74比29.27±0.95,均P<0.01].免疫组化显示,甲减组UCP2蛋白在肾小球和肾小管中的表达均低于对照组(肾小球:0.17±0.02比0.24±0.04,肾小管:0.19±0.02比0.25±0.02,均P<0.01).RT-PCR显示,甲减组UCP2 mRNA表达明显低于对照组(0.70±0.19比1.30±0.09,P<0.01). 结论 甲状腺激素减少时肾脏中UCP2表达下调,提示可能会有损伤肾脏,其机制与肾脏线粒体UCP2表达下调有关.     

解偶联蛋白2在SD大鼠肝缺血再灌注损伤中的表达及意义

目的:探讨解偶联蛋白2(UCP2)在肝缺血再灌注损伤中的作用。方法:选雄性SD大鼠50只,随机分为假手术组(SO组)、30min缺血组(30minI组)、30min缺血再灌注组(30minI/R组)、60min缺血组(60minI组)、60min缺血再灌注组(60minI/R组),每组10只。观察肝组织病理学变化,检测其ALT、ATP及UCP2的表达。结果:SO组、I组和I/R组的血清ALT和肝组织UCP2的表达依次升高,而ATP依次降低;析因方差分析显示缺血、再灌注两因素都使其升高或降低,且有协同作用。病理结果示缺血组肝组织呈灰白色,病理切片可见散在的凋亡细胞;缺血再灌注组呈暗红色,30minI/R组病理切片可见点状坏死,而60minI/R组可见桥接坏死。结论:UCP2在正常肝组织中已有表达;缺血后肝组织中UCP2的表达已经开始升高,ATP减少;缺血再灌注后肝组织UCP2的表达继续升高,ATP进一步减少;UCP2表达升高对肝缺血再灌注损伤有害。     

氯胺酮对大鼠脑缺血后解偶联蛋白2的影响

目的 探讨氧胺酮对大鼠前脑缺血-再灌注(I/R)后海马解偶联蛋白2(uncoupling proteins 2,UCP 2)表达的影响及在脑保护作用中的机制. 方法 45只体质量在250～300 g的雄性Wistar大鼠随机分为3组,每组15只.双侧颈总动脉夹闭加放血降压再回输法建立前脑缺血-再灌注模型,大鼠脑电图出现低幅持续性7 Hz、30～40μV的θ节律为脑缺血模型成功标志.对照组(C组):仅暴露双侧颈总动脉,未放血降压及夹闭双侧颈总动脉;缺血损伤组(1组):放血法使平均动脉压降到(40±5)mmHg时,夹闭双侧颈总动脉10 min,侧脑室内注射生理盐水(1.0 mg/kg);氯胺酮干预组(K组):放血降压、夹闭双侧颈总动脉与Ⅰ组相同,侧脑室内注射氯胺酮(1.0 mg/kg).于再灌注6 h后断头取脑组织,光镜下观察脑组织病理学改变;半定量逆转录.聚合酶链反应(RT-PCR)技术检测海马UCP 2mRNA表达(n=7);免疫组织化学(IH)法检测海马UCP2蛋白表达(n=8).RT-PCR、IH结果应用方差分析(ANOVA)进行统计学分析. 结果 与C组UCP2 mRNA(0,91±0.02)表达比较,Ⅰ组脑I/R后6h UCP2 mRNA(1.06±0.02)和K组脑I/R后6 h UCP2 mRNA(1.18±0.06)表达升高(P<0.05),K组脑I/R后6 h UCP 2 mRNA表达高于Ⅰ组(P<0.05);C组UCP2蛋白少量表达(17.91±5.49),Ⅰ组脑I/R后6 h UCP2蛋白(31.56±4.01)和K组脑I/R后6 h UCP2蛋白(44.61±4.96)表达升高(P<0.05).K组脑I/R后6 h UCP2蛋白表达高于Ⅰ组(P<0.05). 结论 氯胺酮促进大鼠前脑缺血-再灌注(I/R)后海马UCP 2 mRNA和蛋白的表达,可能是其对脑缺血-再灌注损伤保护作用的机制之一.     

解偶联蛋白2表达对颗粒细胞抗氧化应激的影响

目的：通过观察体外培养颗粒细胞解偶联蛋白2（UCP2）表达变化对细胞抗氧化能力的影响，探讨颗粒细胞参与卵泡抗氧化应激的机制.方法：体外培养人颗粒细胞，分别加入过氧化氢（0.25 mmol/L），ATP（1 mmol/L），ATP＋过氧化氢，继续培养24 h，检测颗粒细胞凋亡发生率.结果：过氧化氢可以诱导颗粒细胞UCP2表达，应用ATP抑制UCP2表达后，细胞凋亡发生率增加，细胞抗氧化能力下降.结论：UCP2通过负反馈机制控制细胞内活性氧水平，参与颗粒细胞抗凋亡和对卵母细胞氧化应激保护作用.     

Increased uncoupling protein-2 and -3 mRNA expression during fasting in obese and lean humans.

Uncoupling protein-2 and -3 (UCP2 and UCP3) are mitochondrial proteins that show high sequence homology with the brown adipocyte-specific UCP1. UCP1 induces heat production by uncoupling respiration from ATP synthesis. UCP2 is widely expressed in human tissues, whereas UCP3 expression seems restricted to skeletal muscle, an important site of thermogenesis in humans. We have investigated the regulation of UCP2 and UCP3 gene expression in skeletal muscle and adipose tissue from lean and obese humans. UCP2 and -3 mRNA levels were not correlated with body mass index (BMI) in skeletal muscle, but a positive correlation (r = 0.55, P < 0.01, n = 22) was found between UCP2 mRNA level in adipose tissue and BMI. The effect of fasting was investigated in eight lean and six obese subjects maintained on a hypocaloric diet (1,045 kJ/d) for 5 d. Calorie restriction induced a similar 2-2.5-fold increase in UCP2 and -3 mRNA levels in lean and obese subjects. To study the effect of insulin on UCP gene expression, six lean and five obese subjects underwent a 3-h euglycemic hyperinsulinemic clamp. Insulin infusion did not modify UCP2 and -3 mRNA levels. In conclusion, the similar induction of gene expression observed during fasting in lean and obese subjects shows that there is no major alteration of UCP2 and -3 gene regulation in adipose tissue and skeletal muscle of obese subjects. The increase in UCP2 and -3 mRNA levels suggests a role for these proteins in the metabolic adaptation to fasting.     

Functional polymorphisms of UCP2 and UCP3 are associated with a reduced prevalence of diabetic neuropathy in patients with type 1 diabetes

OBJECTIVE: We studied the association between polymorphisms in the UCP genes and diabetes complications in patients with type 1 diabetes. RESEARCH DESIGN AND METHODS: We analyzed 227 patients with type 1 diabetes using PCR and subsequent cleavage by restriction endonucleases for the promoter variants A-3826G in the UCP1 gene, G-866A in the UCP2 gene, and C-55T in the UCP3 gene. RESULTS: No effect of the A-3826G polymorphism in the UCP1 gene on diabetes complications was found. Patients who were heterozygous or homozygous for the G-866A polymorphism in the UCP2 gene or the C-55T polymorphism in the UCP3 gene had a significantly reduced prevalence of diabetic neuropathy (UCP2: odds ratio 0.44 [95% CI 0.24-0.79], P = 0.007; UCP3: 0.48 [0.25-0.92], P = 0.031), whereas there was no association with other diabetes complications. This effect was stronger when G-866A and C-55T occurred in a cosegregatory manner (UCP2 and UCP3: 0.28 [0.12-0.65], P = 0.002). Furthermore, a multiple logistic regression model showed an age- and diabetes duration-independent effect of the cosegregated polymorphisms on the prevalence of diabetic neuropathy (P = 0.013). CONCLUSIONS: Our data indicate that both the G-866A polymorphism in the UCP2 gene and the C-55T polymorphism in the UCP3 gene are associated with a reduced risk of diabetic neuropathy in type 1 diabetes. Thus, the results presented here support the hypothesis that higher expression of uncoupling protein might prevent mitochondria-mediated neuronal injury and, ultimately, diabetic neuropathy.     

Superoxide-mediated activation of uncoupling protein 2 causes pancreatic beta cell dysfunction

Failure to secrete adequate amounts of insulin in response to increasing concentrations of glucose is an important feature of type 2 diabetes. The mechanism for loss of glucose responsiveness is unknown. Uncoupling protein 2 (UCP2), by virtue of its mitochondrial proton leak activity and consequent negative effect on ATP production, impairs glucose-stimulated insulin secretion. Of interest, it has recently been shown that superoxide, when added to isolated mitochondria, activates UCP2-mediated proton leak. Since obesity and chronic hyperglycemia increase mitochondrial superoxide production, as well as UCP2 expression in pancreatic beta cells, a superoxide-UCP2 pathway could contribute importantly to obesity- and hyperglycemia-induced beta cell dysfunction. This study demonstrates that endogenously produced mitochondrial superoxide activates UCP2-mediated proton leak, thus lowering ATP levels and impairing glucose-stimulated insulin secretion. Furthermore, hyperglycemia- and obesity-induced loss of glucose responsiveness is prevented by reduction of mitochondrial superoxide production or gene knockout of UCP2. Importantly, reduction of superoxide has no beneficial effect in the absence of UCP2, and superoxide levels are increased further in the absence of UCP2, demonstrating that the adverse effects of superoxide on beta cell glucose sensing are caused by activation of UCP2. Therefore, superoxide-mediated activation of UCP2 could play an important role in the pathogenesis of beta cell dysfunction and type 2 diabetes.     

Effects of ethanol on the induction of uncoupling protein-1 (UCP1) mRNA in the mouse brown adipose tissue

Expression of uncoupling protein-1 (UCP1) is increased by cold acclimation and overfeeding, and reduced in fasting and genetic obesity. It is known that the mitochondrial UCP1 in the brown adipose tissue (BAT) is an important key molecule for non-shivering thermogenesis. On the other hand, ethanol (EtOH) alters thermoregulation in humans and laboratory animals. However, the relationship between EtOH intake and UCP1 expression is not yet clear. Accordingly, the present study employed the technique of real-time quantitative polymerase-chain reaction (PCR) to investigate the effects of EtOH (0.5 or 2.0 g/kg) on the expression of UCP1 mRNA in the mouse BAT. Control mice were injected with the same volume of physiological saline intraperitoneally (IP). IP injection of EtOH (0.5 g/kg) caused a decrease and an increase of the expression of BAT UCP1 mRNA at 1 and 4 hours, respectively. Treatment with EtOH (2.0 g/kg) caused an increases of the expression of BAT UCP1 mRNA at both 2 and 4 hours. BAT UCP1 mRNA levels in both groups increased at 4 hours after EtOH administration. The levels of UCP1 mRNA returned to the control levels by 8 hours after EtOH administration. The expression of BAT UCP1 mRNA was upregulated following EtOH administration, although a lower dose of EtOH initially reduced the expression of UCP1 mRNA in BAT. These findings suggest that EtOH-induced UCP1 mRNA expression in BAT reflects an alteration of the set point of thermogenesis.     

Intracellular conversion of thyroxine to triiodothyronine is required for the optimal thermogenic function of brown adipose tissue.

The effect of thyroxine (T4) and triiodothyronine (T3) on the expression of uncoupling protein (UCP) in rat brown adipose tissue (BAT) has been examined. Thyroidectomized rats have a threefold reduction in basal UCP levels. When exposed to cold, they become hypothermic and show a fivefold lower response of UCP than euthyroid controls. T3 augments the basal levels and the response of UCP and its mRNA to cold in a dose-dependent manner. However, to normalize the response of UCP, T3 has to be given in a dosage that produces systemic hyperthyroidism. Mere T3 replacement corrects the systemic hypothyroidism but not the hypothermia or the low levels of UCP. In contrast, replacement doses of T4 prevent the hypothermia and correct the UCP level. Both effects of T4 are blocked by preventing T4 to T3 conversion in BAT. Thus, the optimal UCP response to cold and protection against hypothermia require a high BAT T3 concentration, which is attained from euthyroid levels of T4 via the activation of the BAT T4 5'deiodinase during cold exposure, but not from euthyroid plasma T3 levels.     

Mutations in UCP2 in Congenital Hyperinsulinism Reveal a Role for Regulation of Insulin Secretion

Although the most common mechanism underlying congenital hyperinsulinism is dysfunction of the pancreatic ATP-sensitive potassium channel, the pathogenesis and genetic origins of this disease remains largely unexplained in more than half of all patients. UCP2 knockout mice exhibit an hyperinsulinemic hypoglycemia, suggesting an involment of UCP2 in insulin secretion. However, a possible pathogenic role for UCP2 protein in the development of human congenital hyperinsulinism or of any human disease has not yet been investigated. We studied ten children exhibiting congenital hyperinsulinism, without detectable mutations in the known congenital hyperinsulinism-causing genes. Parental-inherited heterozygous UCP2 variants encoding amino-acid changes were found in two unrelated children with congenital hyperinsulinism. Functional assays in yeast and in insulin-secreting cells revealed an impaired activity of UCP2 mutants. Therefore, we report the finding of UCP2 coding variants in human congenital hyperinsulinism, which reveals a role for this gene in the regulation of insulin secretion and glucose metabolism in humans. Our results show for the first time a direct association between UCP2 amino acid alteration and human disease and highlight a role for mitochondria in hormone secretion.     

解偶联蛋白2启动子区-866G/A多态性与中国北方人肥胖的关系

目的：研究UCP2基因启动子区-866G/A的多态性与中国北方人肥胖的关系.方法：采用PCR及PCR-RFLP方法检测101名正常人的UCP2基因启动子区-866G/A的多态性，测定空腹血糖、胰岛素、血脂、体重及身高，计算体质指数（BMI）.结果：（1）-866G/A基因型频率在中国北方正常人群的分布为，GG 31例（30.7%）、GA 46例（45.5%）、AA 24例（23.8%）.（2）在正常组及超重肥胖组-866G/A的三种基因型频率分布无明显差异.（3）-866G/A多态性的三种基因型的BMI与肥胖相关的表型空腹血糖、空腹胰岛素、血脂差异均无显著性.结论：（1）中国北方人群的UCP2基因启动子区-866G/A基因型、分布与日本人相似，与欧洲白人及高加索人群差异存在显著性.（2）未发现UCP2基因启动子区-866G/A多态性与中国北方人肥胖的关系.     

Endogenous regulation of insulin secretion by UCP2

Uncoupling protein 2 (UCP2) expression is more-or-less ubiquitous, in tissues of diverse function. Its presence in adipose and muscle is postulated to be involved in regulation of energy expenditure and nutrient partitioning, particularly that of fats. In pancreatic islet beta cells, induction of UCP2 is shown to inhibit glucose-stimulated insulin secretion. Thus, insufficient insulin secretion in models of type 2 diabetes is associated with elevated UCP2 expression in islets. The evidence for such a role for UCP2 in the regulation of insulin secretion in islets is reviewed.