【作者】 陈娜；

【导师】 熊燕；

【作者基本信息】 中南大学 ， 药理学， 2007， 硕士

【摘要】 研究背景和目的:胰岛素抵抗和β细胞功能障碍是糖尿病的两大重要病理基础;这两者的发生发展与线粒体功能障碍密切相关。大量研究表明,胰岛素抵抗的小鼠、1型及2型糖尿病动物和患者均伴有骨骼肌、肝脏和脂肪组织中线粒体氧化磷酸化活性下降,ATP生成减少,线粒体功能障碍。线粒体最主要的功能是合成ATP,线粒体ATP的生成不仅由线粒体呼吸酶如琥珀酸脱氢酶和细胞色素C氧化酶活性以及线粒体膜电位决定,还与线粒体生物合成密切相关。过氧化物酶体增殖物激活受体γ辅激活因子1α(PGC-1α)是调节线粒体生物合成的关键基因;此外,线粒体基因如细胞色素C氧化酶亚单位Ⅰ(COXⅠ)与核基因如β-actin的比值亦可用来表示线粒体DNA的相对含量,间接反映线粒体生物合成。近年来发现,解偶联蛋白2(UCP2)在线粒体呼吸链氧化磷酸化解偶联中起重要作用,是线粒体ATP生成减少的另一重要原因。UCP2通过介导质子跨膜内流,驱散线粒体内膜的H~+梯度,降低线粒体膜电位,使ADP磷酸化为ATP所需的跨膜电位势能减少,进而使ATP生成减少,线粒体功能受损。此外,氧化应激也与线粒体功能障碍密切相关,因为线粒体不仅是活性氧簇产生的主要部位,而且是活性氧簇攻击的首要靶点。超氧化物也可通过刺激UCP2的表达,使质子渗漏作用增强,加剧线粒体功能障碍。我室和国内外研究表明,内源性一氧化氮合酶(NOS)抑制物——非对称性二甲基精氨酸(ADMA)升高在胰岛素抵抗、糖尿病以及糖尿病血管并发症中起重要作用。内源性ADMA不仅可抑制NOS活性,减少一氧化氮(NO)生成;还可使NOS解偶联,促进超氧化物生成。最近有研究报道,外源性NOS抑制剂L-N-硝基精氨酸甲酯(L-NAME)可进一步增加花生四烯酸诱导的大鼠肝细胞线粒体膜电位下降,而用一氧化氮(NO)供体S-亚硝基乙酰青霉胺(SNAP)孵育则相反;神经型NOS(nNOS)缺失的小鼠脑、骨骼肌和心肌组织中细胞色素C氧化酶和琥珀酸脱氢酶的活性均明显降低。由此提示NO在线粒体功能调节中的重要作用。基于ADMA是体内NO合成的主要抑制物、NO与线粒体功能障碍有关以及线粒体功能障碍和ADMA在糖尿病中的重要作用,我们推测ADMA很可能通过抑制NOS活性,降低NO生成或增加氧自由基生成,干扰线粒体功能,促进糖尿病及其血管并发症的发生、发展。因此,本课题以糖尿病大鼠肝脏和培养的大鼠肝细胞系(H4IIE)作为研究对象,探讨ADMA在糖尿病大鼠肝脏线粒体功能障碍中的作用;为阐明糖尿病的发病机制提供新的实验证据,为糖尿病临床防治开拓新思路。方法:①动物实验:采用一次性腹腔注射大剂量链脲佐菌素(60mg/kg)诱导1型糖尿病大鼠模型;采用高脂饲养4周加小剂量链脲佐菌素(35mg/kg)腹腔注射的方法制备2型糖尿病大鼠模型,继续喂养8周后,检测两型糖尿病大鼠的血糖以及2型糖尿病大鼠血中胰岛素水平和胰岛素敏感性指数等来评价糖尿病模型;用高效液相法检测血清ADMA浓度;用比色法测定糖尿病大鼠肝脏中线粒体琥珀酸脱氢酶及细胞色素C氧化酶活性,荧光分光光度法检测线粒体膜电位,生物发光法测定ATP含量等指标来评价线粒体功能;并对血清ADMA水平与反映线粒体功能的四个指标进行相关性分析。用逆转录-聚合酶链反应(RT-PCR)测定肝脏PGC-1α基因的mRNA水平及用PCR方法检测线粒体基因COXⅠ与核基因β-actin的拷贝数比值来反映线粒体的生物合成;用硫代巴比妥酸法测定大鼠肝脏丙二醛(MDA)含量及用黄嘌呤氧化酶法检测肝脏超氧化物歧化酶(SOD)活性以反映体内脂质过氧化及抗氧化水平;用硝酸还原酶法检测大鼠肝脏NO含量;用比色法测定肝脏NOS活性;②细胞实验:采用外源性ADMA(30μmol/L)孵育大鼠肝细胞系H4IIE细胞48小时后,观察ADMA对细胞线粒体功能和线粒体生物合成的影响;此外,还用RT-PCR测定解偶联蛋白2(UCP2)基因的转录水平,并检测细胞NO含量、NOS活性,MDA水平以及SOD活性;以探讨ADMA引起线粒体功能障碍的可能机制。结果:①动物实验表明,链脲佐菌素(60 mg/kg)诱导的1型糖尿病大鼠血糖水平明显升高;高脂饲养加链脲佐菌素(35 mg/kg)诱导的2型糖尿病大鼠血糖、胰岛素水平显著升高,胰岛素敏感性指数明显降低,表明1型及2型糖尿病大鼠模型建立成功。饲养8周后,1型及2型糖尿病大鼠血清内源性ADMA浓度显著升高(P＜0.01),并伴有肝脏线粒体琥珀酸脱氢酶(P＜0.01)及细胞色素C氧化酶活性(P＜0.05)明显下降,线粒体膜电位降低(P＜0.05),ATP生成减少(P＜0.01);经相关性分析发现,血清ADMA浓度与反映线粒体功能的四个指标呈显著负相关(P＜0.05)。此外,肝中脂质过氧化产物MDA含量明显增加,SOD活性显著下降(P＜0.01);肝脏NO含量及NOS活性明显降低(P＜0.01)。②细胞实验发现,用1～30μmol/L ADMA孵育大鼠肝细胞H4IIE 12～48h,呈剂量和时间依赖性降低细胞线粒体琥珀酸脱氢酶活性,尤其以30μmol/L ADMA孵育细胞48 h时酶活性降低最明显。因此,在后续的实验中,我们采用30μmol/L ADMA孵育H4IIE细胞48 h,结果显示ADMA显著抑制细胞线粒体琥珀酸脱氢酶和细胞色素C氧化酶活性、降低线粒体膜电位、减少ATP生成,并上调UCP2的mRNA表达;另外,ADMA也下调PGC-1α的mRNA水平,减少线粒体DNA含量,抑制线粒体生物合成。此外,ADMA还抑制细胞NOS活性,减少NO生成,并升高细胞培养液中脂质过氧化产物MDA的含量、降低SOD活性。30μmol/L的L-NAME具有与ADMA类似的作用;而用NO供体硝普钠或抗氧化剂PDTC预孵育1h可对抗ADMA所致的上述作用。结论:本研究结果表明①1型和2型糖尿病大鼠肝脏线粒体功能障碍与血清内源性ADMA浓度升高密切相关;②用外源性ADMA孵育培养的大鼠肝细胞,可直接引起线粒体功能障碍;③ADMA导致线粒体功能障碍可能与减少NO生成,增加氧化应激;上调UCP2的转录表达;下调PGC-1αmRNA水平,抑制线粒体生物合成等有关。更多还原

【Abstract】 BACKGROUND Insulin resistance and pancreasticβcell dysfunction are two primary pathologyical basis of diabetes mellitus,and these prominent features of diabetes are attributable to mitochondrial dysfunction. Accumulation evidence showed that mitochondrial dysfunction of skeletal muscle,liver or adipose tissue were found in insulin resistant mice,animals and patients with type 1 or type 2 diabetes.The most crucial task of mitochondria is the generation of energy in the form of ATP,the generation of ATP in mitochondria is not only determined by the activities of respiratory enzymes such as succinate dehydrogenase and cytochrome C oxidase as well as mitochondrial transmembrane potential,but also closely related to mitochondrial biogenesis.Peroxisome proliferators-activated receptorγcoactivator-1α(PGC-1α)is a master regulator of mitochondrial biogenesis; Furthermore,the copy number ratio of mitochondrial genes such as cytochrome C oxidase subunitⅠ(COXⅠ)and nuclear genes likeβ-actin is often used to reflect mitochondrial biogenesis indirectly.Recent studies have shown that uncoupling protein 2(UCP2)plays an important role in uncoupling oxidation and phosphorylation in mitochondria.UCP2 mediated protons leaking across the mitochondrial inner membrane,lowered mitochondrial membrane potential,hence uncoupled glucose oxidative metabolism from ATP production,impaired mitochondrial function.In addition,oxidative stress is also associated with mitochondrial dysfunction,since mitochondria is not only a major source of reactive oxygen species,but also the first target for attacking of reactive oxygen species.Stimulation of UCP2 activity by superoxed is also relevant to the development of mitochondrial dysfunction.A series of experiments showed that the elevation of endogenous nitric oxide synthase(NOS)inhibitor asymmetric dimethylarginine(ADMA)plays important roles in insulin resistance,diabetes and diabetic vascular complications.Endogenous ADMA not only can inhibit NOS activity reducing NO synthesis;but also can uncouple NOS generating superoxide. Recent studies have shown that L-N(G)-Nitroarginine Methylester(L-NAME) increases arachidonic acid-induced decline of mitochondrial membrane potential in rat primary hepatocytes,but NO donor S-nitrosoacetyl penicillamine(SNAP)has an opposite effect.The activities of cytochrome C oxidase and succinate dehydrogenase are impaired in the brain,muscle and heart of mice lacking the neuronal nitric oxide synthase isoform(nNOS-KO). Therefore,NO has been considered as a new key molecule in maintenance of mitochondrial function.Since ADMA is a critical inhibitor of NOS,NO is associated with mitochondrial dysfunction,and mitochondrial dysfunction as well as ADMA play important roles in diabetes mellitus,we presumed that ADMA might impaire mitochondrial function,contribute to the development of diabetes and its vascular complications via inhibiting NOS activity, decreasing NO generation or enhancing oxidative stress.Accordingly,the present study was designed to examine the alterations in serum ADMA concentrations and liver mitochondrial function of type 1 and type 2 diabtic rats,and to explore the effects of ADMA on mitochondrial function in rat hepatoma carcinoma cell H4IIE.These results may provide novel experimental evidence for the role of ADMA in the development of mitochondrial dysfunction and its potential mechanisms,and further provide new insight into the clinical prevention and treatment of diabetes mellitus.METHODS①In animal experiments,type 1 diabetic rats were induced by a single injection of streptozotocin(60 mg/kg,i.p.),and type 2 diabetic rats were induced by first feeding high-fat diet to animal for 4 weeks and second injecting streptozotocin(35 mg/kg,i.p.)to rats in bolus.After 8 weeks,blood levels of glucose were determined.Serum ADMA was analysed by high performance liquid chromatography.Liver mitochondrial succinate dehydrogenase and cytochrome C oxidase activities were measured by colorimetry,mitochondrial transmembrane potential was detected by fluorospectrophotometry,and ATP levels were assessed by luciferin/luciferase reaction.Malondialdehyde(MDA)contents and superoxide dismutase activity were measured by colorimetry to reflect the lipid peroxidation and antioxidant ability,respectively.①In cell experiments,rat hepatoma carcinoma cells (H4IIE)were incubated with 30μmol/L ADMA for 48 hours in the absence or presence of exogenous NOS inhibitor L-NAME(10μmol/L),NO donor sodium nitroprusside or antioxidant pyrrolidine dithiocarbamate(PDTC). Mitochondrial succinate dehydrogenase and cytochrome C oxidase activities, mitochondrial transmembrane potential and ATP contents were examined as mentioned above.Reverse transcription-polymerase chain reaction(RT-PCR) was used to detect the mRNA levels of PGC-1α,and polymerase chain reaction(PCR)was used to assesse the copy numbers of mitochondrial genes such as COXⅠand nuclear genes such asβ-actin,these indexes were used to reflect mitochondrial biogenesis.In addition,RT-PCR was also used to measure the mRNA expression of uncoupling protein 2(UCP2)to evaluate mitochondrial oxidative phosphorylation uncoupling condition.NOS activity and NO concentrations,MDA contents and SOD activity were assayed to illuminate the possible mechanism of mitochondrial dysfunction induced by ADMA.RESULTS①In animal experiments,serum ADMA levels were markedly elevated(P＜0.01),accompanied by impaired mitochondrial succinate dehydrogenase(P＜0.01)and cytochrome C oxidase(P＜0.01)activities, lowered mitochondrial transmembrane potential(P＜0.05)and decreased ATP levels(P＜0.01)in the liver from.type 1 and type 2 diabetic rats compared to control rats.In addition,MDA contents were elevated significantly while SOD activity was reduced markedly in diabetic rats liver.②In cell experiments, H4IIE hepatocytes were incubated with 1～30μmol/L ADMA for 12～48 h, mitochondrial succinate dehydrogenase activity was inhibited in a dose-and time-dependent manner,indicating that succinate dehydrogenase activity was impaired in H4IIE cells especially after exposure to 30μmol/L ADMA for 48 h.Accordingly,we chose 30μmol/L ADMA and 48 h in our following experiments.We found that ADMA markedly inhibited mitochondrial function characterized by reduced succinate dehydrogenase activity,lowered mitochondrial transmembrane potential and declined ATP contents,besides, up-regulated UCP2 mRNA levels;It also down-regulated the expression of PGC-1α,reduced mitochondrial DNA contents,inhibited mitochondrial biogenesis.Moreover,ADMA inhibited NOS activity,decreased NO contents, increased MDA levels and significantly impaired SOD activities.Similar effect was observed when cells were treated with 30μmol/L L-NAME. Preincubation with sodium nitroprusside or PDTC reversed these effects of ADMA on H4IIE hepatocytes.CONCLUSIONS①Mitochondrial dysfunction in liver of diabetic rats was closely related to elevated endogenous inhibitor of nitric oxide synthase ADMA in serum;②In cultured H4IIE hepatocytes,exogenous ADMA could directly impaire mitochondrial function;③The mechanisms underlying which ADMA induced mitochondrial dysfuction could be related to the decrease of NO production and increase of oxidative stress via uncoupling NOS,upgulating UCP2 transcription,and downregulating PGC-1αtranscription.更多还原