非对称性二甲基精氨酸与糖尿病大鼠肝脏线粒体功能障碍
|
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。
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.
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。
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.
引文
1 Zimmet P, Alberti KG, Shaw J. Global and societal implications of the diabetes epidemic. Nature, 2001,414:782-7.
2 Cheng TO. Diabetes and obesity epidemics in China: A national crisis. Int J Cardiol, 2007.
3 Lowell BB, Shulman GI. Mitochondrial dysfunction and type 2 diabetes. Science, 2005,307:384-7.
4 Itani SI, Ruderman NB, Schmieder F, et al. Lipid-induced insulin resistance in human muscle is associated with changes in diacylglycerol, protein kinase C, and IkappaB-alpha. Diabetes, 2002,51:2005-11.
5 Langin D. Diabetes, insulin secretion, and the pancreatic beta-cell mitochondrion. N Engl J Med, 2001,345:1772-4.
6 Chan CB, MacDonald PE, Saleh MC, et al. Overexpression of uncoupling protein 2 inhibits glucose-stimulated insulin secretion from rat islets. Diabetes, 1999,48:1482-6.
7 Boudina S, Sena S, O'Neill BT, et al. Reduced mitochondrial oxidative capacity and increased mitochondrial uncoupling impair myocardial energetics in obesity. Circulation, 2005,112:2686-95.
8 Petersen KF, Befroy D, Dufour S, et al. Mitochondrial dysfunction in the elderly: possible role in insulin resistance. Science, 2003,300:1140-2.
9 Petersen KF, Dufour S, Befroy D, et al. Impaired mitochondrial activity in the insulin-resistant offspring of patients with type 2 diabetes. N Engl J Med, 2004,350:664-71.
10 Ho JK, Duclos RI Jr, Hamilton JA. Interactions of acyl carnitines with model membranes: a (13)C-NMR study. J Lipid Res, 2002,43:1429-39.
11 Echtay KS, Murphy MP, Smith RA, et al. Superoxide activates mitochondrial uncoupling protein 2 from the matrix side. Studies using targeted antioxidants. J Biol Chem, 2002,277:47129-35.
12 Kelley DE, He J, Menshikova EV, et al. Dysfunction of mitochondria in human skeletal muscle in type 2 diabetes. Diabetes, 2002,51:2944-50.
13 Hojlund K, Wrzesinski K, Larsen PM, et al. Proteome analysis reveals phosphorylation of ATP synthase beta -subunit in human skeletal muscle and proteins with potential roles in type 2 diabetes. J Biol Chem, 2003,278:10436-42.
14 Choo HJ, Kim JH, Kwon OB, et al. Mitochondria are impaired in the adipocytes of type 2 diabetic mice. Diabetologia, 2006,49:784-91.
15 Ferreira FM, Palmeira CM, Seica R, et al. Diabetes and mitochondrial bioenergetics: alterations with age. J Biochem Mol Toxicol, 2003,17:214-22.
16 Brownlee M. A radical explanation for glucose-induced beta cell dysfunction. J Clin Invest, 2003,112:1788-90.
17 Alano CC, Beutner G, Dirksen RT, et al. Mitochondrial permeability transition and calcium dynamics in striatal neurons upon intense NMDA receptor activation. J Neurochem, 2002,80:531-8.
18 Moreira PI, Santos MS, Sena C, et al. CoQ10 therapy attenuates amyloid beta-peptide toxicity in brain mitochondria isolated from aged diabetic rats. Exp Neurol, 2005,196:112-9.
19 Fukumura A, Tsutsumi M, Tsuchishima M, et al. Correlation between adenosine triphosphate content and apoptosis in liver of rats treated with alcohol. Alcohol Clin Exp Res, 2003,27:12S-5S.
20 Lehman JJ, Barger PM, Kovacs A, et al. Peroxisome proliferator-activated receptor gamma coactivator-1 promotes cardiac mitochondrial biogenesis. J Clin Invest, 2000,106:847-56.
21 Irrcher L Adhihetty PJ, Sheehan T, et al. PPARgamma coactivator-1 alpha expression during thyroid hormone- and contractile activity-induced mitochondrial adaptations. Am J Physiol Cell Physiol, 2003,284:C1669-77.
22 Stuhlinger MC, Abbasi F, Chu JW, et al. Relationship between insulin resistance and an endogenous nitric oxide synthase inhibitor. JAMA, 2002,287:1420-6.
23 Xiong Y, Fu YF, Fu SH, et al. Elevated levels of the serum endogenous inhibitor of nitric oxide synthase and metabolic control in rats with streptozotocin-induced diabetes. J Cardiovasc Pharmacol, 2003,42:191-6.
24 Xiong Y, Lu R, Li YJ, et al. Elevation of an endogenous inhibitor of nitric oxide synthase in diabetic rat serum. Zhongguo Yao Li Xue Bao, 1997,18:511-4.
25 Asagami T, Abbasi F, Stuelinger M, et al. Metformin treatment lowers asymmetric dimethylarginine concentrations in patients with type 2 diabetes. Metabolism, 2002,51:843-6.
26 Xiong Y, Lei M, Fu S, et al. Effect of diabetic duration on serum concentrations of endogenous inhibitor of nitric oxide synthase in patients and rats with diabetes. Life Sci, 2005,77:149-59.
27 Krzyzanowska K,Mittermayer F,Wolzt M,et al.Asymmetric dimethylarginine predicts cardiovascular events in patients with type 2 diabetes.Diabetes Care,2007.
28 Vallance P,Leone A,Calver A,et al.Endogenous dimethylarginine as an inhibitor of nitric oxide synthesis.J Cardiovasc Pharmacol,1992,20 Suppl 12:S60-2.
29 Boger RH,Bode-Boger SM,Tsao PS,et al.An endogenous inhibitor of nitric oxide synthase regulates endothelial adhesiveness for monocytes.J Am Coll Cardiol,2000,36:2287-95.
30 Marui N,Offermann MK,Swerlick P,,et al.Vascular cell adhesion molecule-1(VCAM-1)gene transcription and expression are regulated through an antioxidant-sensitive mechanism in human vascular endothelial cells.J Clin Invest,1993,92:1866-74.
31 Wu D,Cederbaum A.Nitric oxide donors prevent while the nitric oxide synthase inhibitor L-NAME increases arachidonic acid plus CYP2E1-depeadent toxicity.Toxicol Appl Pharmacol,2006,216:282-92.
32 Schild L,Jaroscakova I,Lendeckel U,et al.Neuronal nitric oxide synthase controls enzyme activity pattern of mitochondria and lipid metabolism.FASEB J,2006,20:145-7.
33 Lin KY,Ito A,Asagami T,et al.Impaired nitric oxide synthase pathway in diabetes mellitus:role of asymmetric dimethylarginine and dimethylarginine dimethylaminohydrolase.Circulation,2002,106:987-92.
34 Luo J,Quan J,Tsai J,et al.Nongenetic mouse models of non-insulin-dependent diabetes mellitus.Metabolism,1998,47:663-8.
35 Chen BM,Xia LW,Zhao RQ.Determination of N(G),N(G)-dimethylarginine in human plasma by high-performance liquid chromatography.J Chromatogr B Biomed Sci Appl,1997,692:467-71.
36 Morin C,Zini R,Simon N,et al.Dehydroepiandrosterone and alpha-estradiol limit the functional alterations of rat brain mitochondria submitted to different experimental stresses.Neuroscience,2002,115:415-24.
37 Munujos P,Coll-Canti J,Gonzalez-Sastre F,et al.Assay of succinate dehydrogenase activity by a colorimetric-continuous method using iodonitrotetrazolium chloride as electron acceptor.Anal Biochem,1993,212:506-9.
38 Baracca A,Sgarbi G,Solaini G,et al.Rhodamine 123 as a probe of mitochondrial membrane potential:evaluation of proton flux through F(0)during ATP synthesis.Biochim Biophys Acta,2003,1606:137-46.
39 Scaduto RC Jr, Grotyohann LW. Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives. Biophys J, 1999,76:469-77.
40 Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 1976,72.248-54.
41 Fu YF, Xiong Y, Guo Z. A reduction of endogenous asymmetric dimethylarginine contributes to the effect of captopril on endothelial dysfunction induced by homocysteine in rats. Eur J Pharmacol, 2005,508:167-75.
42 Akao Y, Otsuki Y, Kataoka S, et al. Multiple subcellular localization of bcl-2: detection in nuclear outer membrane, endoplasmic reticulum membrane, and mitochondrial membranes. Cancer Res, 1994,54:2468-71.
43 Kibirige M, Metcalf B, Renuka R, et al. Testing the accelerator hypothesis: the relationship between body mass and age at diagnosis of type 1 diabetes. Diabetes Care, 2003,26:2865-70.
44 Lupi R, Marselli L, Dionisi S, et al. Improved insulin secretory function and reduced chemotactic properties after tissue culture of islets from type 1 diabetic patients. Diabetes Metab Res Rev, 2004,20:246-51.
45 Chavin KD, Yang S, Lin HZ, et al. Obesity induces expression of uncoupling protein-2 in hepatocytes and promotes liver ATP depletion. J Biol Chem, 1999,274:5692-700.
46 Stuart JA, Harper JA, Brindle KM, et al. Physiological levels of mammalian uncoupling protein 2 do not uncouple yeast mitochondria. J Biol Chem, 2001,276:18633-9.
47 Chan CB, De Leo D, Joseph JW, et al. Increased uncoupling protein-2 levels in beta-cells are associated with impaired glucose-stimulated insulin secretion: mechanism of action. Diabetes, 2001,50:1302-10.
48 Nagase I, Yoshida T, Saito M. Up-regulation of uncoupling proteins by beta-adrenergic stimulation in L6 myotubes. FEBS Lett, 2001,494:175-80.
49 Zhang CY, Baffy G, Perret P, et al. Uncoupling protein-2 negatively regulates insulin secretion and is a major link between obesity, beta cell dysfunction, and type 2 diabetes. Cell, 2001,105:745-55.
50 Merial C, Bouloumie A, Trocheris V, et al. Nitric oxide-dependent downregulation of adipocyte UCP-2 expression by tumor necrosis factor-alpha. Am J Physiol Cell Physiol, 2000, 279:C1100-6.
51 Fridlyand LE, Philipson LH. Reactive species and early manifestation of insulin resistance in type 2 diabetes.Diabetes Obes Metab,2006,8:1 36-45.
52 Sydow K,Munzel T.ADMA and oxidative stress.Atheroscler Suppl,2003,4:41-51.
53 Ricquier D,Bouillaud F.The uncoupling protein homologues:UCP1,UCP2,UCP3,StUCP and AtUCP.Biochem J,2000,345 Pt 2:161-79.
54 Mohazzab KM,Kaminski PM,Wolin MS.NADH oxidoreductase is a major source of superoxide anion in bovine coronary artery endothelium.Am J Physiol,1994,266:H2568-72.
55 Jia SJ,Jiang DJ,Hu CP,et al.Lysophosphatidylcholine-induced elevation of asymmetric dimethylarginine level by the NADPH oxidase pathway in endothelial cells.Vascul Pharmacol,2006,44:143-8.
56 Cai H,Harrison DG.Endothelial dysfunction in cardiovascular diseases:the role of oxidant stress.Circ Res,2000,87:840-4.
57 Rasbach KA,Schnellmann RG.Signaling of mitochondrial biogenesis following oxidant injury.J Biol Chem,2007,282:2355-62.
58 Brown GC.Cell biology.NO says yes to mitochondria.Science,2003,299:838-9.
59 Nisoli E,.Clementi E,Paolucci C,et al.Mitochondrial biogenesis in mammals:the role of endogenous nitric oxide.Science,2003,299:896-9.
1 Pinhas-Hamiel O, Zeitler P. Acute and chronic complications of type 2 diabetes mellitus in children and adolescents. Lancet, 2007,369:1823-31.
2 Wild S, Roglic G, Green A, et al. Global prevalence of diabetes: estimates for the year 2000 and projections for 2030. Diabetes Care, 2004,27:1047-53.
3 Lowell BB, Shulman GI. Mitochondrial dysfunction and type 2 diabetes. Science, 2005,307:384-7.
4 St John JC, Amaral A, Bowles E, et al. The analysis of mitochondria and mitochondrial DNA in human embryonic stem cells. Methods Mol Biol, 2006,331:347-74.
5 Szewczyk A, Wojtczak L. Mitochondria as a pharmacological target. Pharmacol Rev, 2002,54:101-27.
6 Cohen BH, Gold DR. Mitochondrial cytopathy in adults: what we know so far. Cleve Clin J Med, 2001,68:625-6, 629-42.
7 Heales SJ, Bolanos JP. Impairment of brain mitochondrial function by reactive nitrogen species: the role of glutathione in dictating susceptibility. Neurochem Int, 2002,40:469-74.
8 Brownlee M. A radical explanation for glucose-induced beta cell dysfunction. J Clin Invest, 2003,112:1788-90.
9 Taniguchi A, Fukushima M, Nakai Y, et al. Factors responsible for the evolution of insulin resistance in Japanese type 2 diabetic patients: Association with atherosclerosis. Diabetes Res Clin Pract, 2007.
10 Roos C, Lidfeldt J, Agardh CD, et al. Insulin resistance and self-rated symptoms of depression in Swedish women with risk factors for diabetes: the Women's Health in the Lund Area study. Metabolism, 2007,56:825-9.
11 Dela F. Other adaptations to training/inactivity in type 2 diabetics and other groups with insulin resistance: emphasis on prevention of CHD. Appl Physiol Nutr Metab, 2007,32:602-6.
12 Petersen KF, Dufour S, Befroy D, et al. Impaired mitochondrial activity in the insulin-resistant offspring of patients with type 2 diabetes. N Engl J Med, 2004,350:664-71.
13 Chiasson JL, Brindisi MC, Rabasa-Lhoret R. The prevention of type 2 diabetes: what is the evidence?. Minerva Endocrinol, 2005,30:179-91.
14 Boden G, Shulman GI. Free fatty acids in obesity and type 2 diabetes: defining their role in the development of insulin resistance and beta-cell dysfunction. Eur J Clin Invest, 2002,32 Suppl 3:14-23.
15 Shulman GI. Cellular mechanisms of insulin resistance. J Clin Invest, 2000,106:171-6.
16 Yu C, Chen Y, Cline GW, et al. Mechanism by which fatty acids inhibit insulin activation of insulin receptor substrate-1 (IRS-1)-associated phosphatidylinositol 3-kinase activity in muscle. J Biol Chem, 2002,277:50230-6.
17 Kraegen EW, Cooney GJ, Ye JM, et al. The role of lipids in the pathogenesis of muscle insulin resistance and beta cell failure in type II diabetes and obesity. Exp Clin Endocrinol Diabetes, 2001,109 Suppl 2:S189-201.
18 RANDLE PJ, GARLAND PB, HALES CN, et al. The glucose fatty-acid cycle. Its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus. Lancet, 1963,1:785-9.
19 Clerk LH, Rattigan S, Clark MG. Lipid infusion impairs physiologic insulin-mediated capillary recruitment and muscle glucose uptake in vivo. Diabetes, 2002,51:1138-45.
20 Boden G, Chen X. Effects of fat on glucose uptake and utilization in patients with non-insulin-dependent diabetes. J Clin Invest, 1995,96:1261-8.
21 Blaak EE, Wolffenbuttel BH, Saris WH, et al. Weight reduction and the impaired plasma-derived free fatty acid oxidation in type 2 diabetic subjects. J Clin Endocrinol Metab, 2001,86:1638-44.
22 Mensink M, Blaak EE, van Baak MA, et al. Plasma free Fatty Acid uptake and oxidation are already diminished in subjects at high risk for developing type 2 diabetes. Diabetes, 2001,50:2548-54.
23 Noguchi T, Kasuga M. [Insulin action and its regulatory mechanisms]. Nippon Rinsho, 2006,64 Suppl 9:121-5.
24 Ferreira-Maldent N, Maillot F, Quilliet L, et al. [Dilated cardiomyopathy, diabetes and deafness related to a mutation of mitochondrial DNA]. Arch Mal Coeur Vaiss, 2007,100:149-52.
25 Petersen KF, Befroy D, Dufour S, et al. Mitochondrial dysfunction in the elderly: possible role in insulin resistance. Science, 2003,300:1140-2.
26 Trifunovic A, Wredenberg A, Falkenberg M, et al. Premature ageing in mice expressing defective mitochondrial DNA polymerase. Nature, 2004,429:417-23.
27 Boudina S, Sena S, O'Neill BT, et al. Reduced mitochondrial oxidative capacity and increased mitochondrial uncoupling impair myocardial energetics in obesity. Circulation, increased mitochondrial uncoupling impair myocardial energetics in obesity.Circulation,2005,112:2686-95.
28 Kelley DE,He J,Menshikova EV,et al.Dysfunction of mitochondria in human skeletal muscle in type 2 diabetes.Diabetes,2002,51:2944-50.
29 Short KR,Nair KS,Stump CS.Impaired mitochondrial activity and insulin-resistant offspring of patients with type 2 diabetes.N Engl J Med,2004,350:2419-21;author reply 2419-21.
30 Wu Z,Puigserver P,Andersson U,et al.Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1.Cell,1999,98:115-24.
31 Mootha VK,Lindgren CM,Eriksson KF,et al.PGC-lalpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes.Nat Genet,2003,34:267-73.
32 Patti ME,Butte AJ,Crunkhorn S,et al.Coordinated reduction of genes of oxidative metabolism in humans with insulin resistance and diabetes:Potential role of PGC1 and NRF1.Proc Natl Acad Sci U S A,2003,100:8466-71.
33 Irrcher I,Adhihetty PJ,Sheehan T,et al.PPARgamma coactivator-lalpha expression during thyroid hormone- and contractile activity-induced mitochondrial adaptations.Am J Physiol Cell Physiol,2003,284:C 1669-77.
34 Chan CB,De Leo D,Joseph JW,et al.Increased uncoupling protein-2 levels in beta-cells are associated with impaired glucose-stimulated insulin secretion:mechanism of action.Diabetes,2001,50:1302-10.
35 Zhang CY,Baffy G,Perret P,et al.Uncoupling protein-2 negatively regulates insulin secretion and is a major link between obesity,beta cell dysfunction,and type 2diabetes.Cell,2001,105:745-55.
36 Kassis N,Bernard C,Pusterla A,et al.Correlation between pancreatic islet uncoupling protein-2(UCP2)mRNA concentration and insulin status in rats.Int J Exp Diabetes Res,2000,1:185-93.
37 Brown JE,Thomas S,Digby JE,et al.Glucose induces and leptin decreases expression of uncoupling protein-2 mRNA in human islets.FEBS Lett,2002,513:189-92.
38 Krempler F,Esterbauer H,Weitgasser R,et al.A functional polymorphism in the promoter of UCP2 enhances obesity risk but reduces type 2 diabetes risk in obese middle-aged humans.Diabetes,2002,51:3331-5.
39 Sesti G,Cardellini M,Marini MA,et al.A common polymorphism in the promoter of UCP2 contributes to the variation in insulin secretion in glucose-tolerant subjects. Diabetes, 2003,52:1280-3
40 Sasahara M, Nishi M, Kawashima H, et al. Uncoupling protein 2 promoter polymorphism -866G/A affects its expression in beta-cells and modulates clinical profiles of Japanese type 2 diabetic patients. Diabetes, 2004,53:482-5.
41 Mattiasson G, Sullivan PG. The emerging functions of UCP2 in health, disease, and therapeutics. Antioxid Redox Signal, 2006,8:1-38.
42 Kelley DE, Mintun MA, Watkins SC, et al. The effect of non-insulin-dependent diabetes mellitus and obesity on glucose transport and phosphorylation in skeletal muscle. J Clin Invest, 1996,97:2705-13.
43 Szendroedi J, Schmid AI, Chmelik M, et al. Muscle mitochondrial ATP synthesis and glucose transport/phosphorylation in type 2 diabetes. PLoS Med, 2007,4:e154.
44 Kashemsant N, Chan CB. Impact of uncoupling protein-2 overexpression on proinsulin processing. J Mol Endocrinol, 2006,37:517-26.
45 Echtay KS, Murphy MP, Smith RA, et al. Superoxide activates mitochondrial uncoupling protein 2 from the matrix side. Studies using targeted antioxidants. J Biol Chem, 2002,277:47129-35.
46 Krauss S, Zhang CY, Scorrano L, et al. Superoxide-mediated activation of uncoupling protein 2 causes pancreatic beta cell dysfunction. J Clin Invest, 2003,112:1831-42.
47 Langin D. Diabetes, insulin secretion, and the pancreatic beta-cell mitochondrion. N Engl J Med, 2001,345:1772-4.
48 Bindokas VP, Kuznetsov A, Sreenan S, et al. Visualizing superoxide production in normal and diabetic rat islets of Langerhans. J Biol Chem, 2003,278:9796-801.
49 Brownlee M. Biochemistry and molecular cell biology of diabetic complications. Nature, 2001,414:813-20.
50 El Midaoui A, de Champlain J. Prevention of hypertension, insulin resistance, and oxidative stress by alpha-lipoic acid. Hypertension, 2002,39:303-7.
51 Du Y, Miller CM, Kern TS. Hyperglycemia increases mitochondrial superoxide in retina and retinal cells. Free Radic Biol Med, 2003,35:1491-9.
52 Korshunov SS, Skulachev VP, Starkov AA. High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria. FEBS Lett, 1997,416:15-8.
53 Du XL, Edelstein D, Dimmeler S, et al. Hyperglycemia inhibits endothelial nitric oxide synthase activity by posttranslational modification at the Akt site. J Clin Invest, 2001,108 1341-8
54 Victor VM, Rocha M. Targeting antioxidants to mitochondria a potential new therapeutic strategy for cardiovascular diseases. Curr Pharm Des, 2007,13:845-63.
55 Nishikawa T, Edelstein D, Du XL, et al. Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage. Nature, 2000,404:787-90.
56 Du XL, Edelstein D, Rossetti L, et al. Hyperglycemia-induced mitochondrial superoxide overproduction activates the hexosamine pathway and induces piasminogen activator inhibitor-1 expression by increasing Sp1 glycosylation. Proc Natl Acad Sci U S A, 2000,97:12222-6.
57 Du X, Matsumura T, Edelstein D, et al. Inhibition of GAPDH activity by poly(ADP-ribose) polymerase activates three major pathways of hyperglycemic damage in endothelial cells. J Clin Invest, 2003,112:1049-57.
2 Cheng TO. Diabetes and obesity epidemics in China: A national crisis. Int J Cardiol, 2007.
3 Lowell BB, Shulman GI. Mitochondrial dysfunction and type 2 diabetes. Science, 2005,307:384-7.
4 Itani SI, Ruderman NB, Schmieder F, et al. Lipid-induced insulin resistance in human muscle is associated with changes in diacylglycerol, protein kinase C, and IkappaB-alpha. Diabetes, 2002,51:2005-11.
5 Langin D. Diabetes, insulin secretion, and the pancreatic beta-cell mitochondrion. N Engl J Med, 2001,345:1772-4.
6 Chan CB, MacDonald PE, Saleh MC, et al. Overexpression of uncoupling protein 2 inhibits glucose-stimulated insulin secretion from rat islets. Diabetes, 1999,48:1482-6.
7 Boudina S, Sena S, O'Neill BT, et al. Reduced mitochondrial oxidative capacity and increased mitochondrial uncoupling impair myocardial energetics in obesity. Circulation, 2005,112:2686-95.
8 Petersen KF, Befroy D, Dufour S, et al. Mitochondrial dysfunction in the elderly: possible role in insulin resistance. Science, 2003,300:1140-2.
9 Petersen KF, Dufour S, Befroy D, et al. Impaired mitochondrial activity in the insulin-resistant offspring of patients with type 2 diabetes. N Engl J Med, 2004,350:664-71.
10 Ho JK, Duclos RI Jr, Hamilton JA. Interactions of acyl carnitines with model membranes: a (13)C-NMR study. J Lipid Res, 2002,43:1429-39.
11 Echtay KS, Murphy MP, Smith RA, et al. Superoxide activates mitochondrial uncoupling protein 2 from the matrix side. Studies using targeted antioxidants. J Biol Chem, 2002,277:47129-35.
12 Kelley DE, He J, Menshikova EV, et al. Dysfunction of mitochondria in human skeletal muscle in type 2 diabetes. Diabetes, 2002,51:2944-50.
13 Hojlund K, Wrzesinski K, Larsen PM, et al. Proteome analysis reveals phosphorylation of ATP synthase beta -subunit in human skeletal muscle and proteins with potential roles in type 2 diabetes. J Biol Chem, 2003,278:10436-42.
14 Choo HJ, Kim JH, Kwon OB, et al. Mitochondria are impaired in the adipocytes of type 2 diabetic mice. Diabetologia, 2006,49:784-91.
15 Ferreira FM, Palmeira CM, Seica R, et al. Diabetes and mitochondrial bioenergetics: alterations with age. J Biochem Mol Toxicol, 2003,17:214-22.
16 Brownlee M. A radical explanation for glucose-induced beta cell dysfunction. J Clin Invest, 2003,112:1788-90.
17 Alano CC, Beutner G, Dirksen RT, et al. Mitochondrial permeability transition and calcium dynamics in striatal neurons upon intense NMDA receptor activation. J Neurochem, 2002,80:531-8.
18 Moreira PI, Santos MS, Sena C, et al. CoQ10 therapy attenuates amyloid beta-peptide toxicity in brain mitochondria isolated from aged diabetic rats. Exp Neurol, 2005,196:112-9.
19 Fukumura A, Tsutsumi M, Tsuchishima M, et al. Correlation between adenosine triphosphate content and apoptosis in liver of rats treated with alcohol. Alcohol Clin Exp Res, 2003,27:12S-5S.
20 Lehman JJ, Barger PM, Kovacs A, et al. Peroxisome proliferator-activated receptor gamma coactivator-1 promotes cardiac mitochondrial biogenesis. J Clin Invest, 2000,106:847-56.
21 Irrcher L Adhihetty PJ, Sheehan T, et al. PPARgamma coactivator-1 alpha expression during thyroid hormone- and contractile activity-induced mitochondrial adaptations. Am J Physiol Cell Physiol, 2003,284:C1669-77.
22 Stuhlinger MC, Abbasi F, Chu JW, et al. Relationship between insulin resistance and an endogenous nitric oxide synthase inhibitor. JAMA, 2002,287:1420-6.
23 Xiong Y, Fu YF, Fu SH, et al. Elevated levels of the serum endogenous inhibitor of nitric oxide synthase and metabolic control in rats with streptozotocin-induced diabetes. J Cardiovasc Pharmacol, 2003,42:191-6.
24 Xiong Y, Lu R, Li YJ, et al. Elevation of an endogenous inhibitor of nitric oxide synthase in diabetic rat serum. Zhongguo Yao Li Xue Bao, 1997,18:511-4.
25 Asagami T, Abbasi F, Stuelinger M, et al. Metformin treatment lowers asymmetric dimethylarginine concentrations in patients with type 2 diabetes. Metabolism, 2002,51:843-6.
26 Xiong Y, Lei M, Fu S, et al. Effect of diabetic duration on serum concentrations of endogenous inhibitor of nitric oxide synthase in patients and rats with diabetes. Life Sci, 2005,77:149-59.
27 Krzyzanowska K,Mittermayer F,Wolzt M,et al.Asymmetric dimethylarginine predicts cardiovascular events in patients with type 2 diabetes.Diabetes Care,2007.
28 Vallance P,Leone A,Calver A,et al.Endogenous dimethylarginine as an inhibitor of nitric oxide synthesis.J Cardiovasc Pharmacol,1992,20 Suppl 12:S60-2.
29 Boger RH,Bode-Boger SM,Tsao PS,et al.An endogenous inhibitor of nitric oxide synthase regulates endothelial adhesiveness for monocytes.J Am Coll Cardiol,2000,36:2287-95.
30 Marui N,Offermann MK,Swerlick P,,et al.Vascular cell adhesion molecule-1(VCAM-1)gene transcription and expression are regulated through an antioxidant-sensitive mechanism in human vascular endothelial cells.J Clin Invest,1993,92:1866-74.
31 Wu D,Cederbaum A.Nitric oxide donors prevent while the nitric oxide synthase inhibitor L-NAME increases arachidonic acid plus CYP2E1-depeadent toxicity.Toxicol Appl Pharmacol,2006,216:282-92.
32 Schild L,Jaroscakova I,Lendeckel U,et al.Neuronal nitric oxide synthase controls enzyme activity pattern of mitochondria and lipid metabolism.FASEB J,2006,20:145-7.
33 Lin KY,Ito A,Asagami T,et al.Impaired nitric oxide synthase pathway in diabetes mellitus:role of asymmetric dimethylarginine and dimethylarginine dimethylaminohydrolase.Circulation,2002,106:987-92.
34 Luo J,Quan J,Tsai J,et al.Nongenetic mouse models of non-insulin-dependent diabetes mellitus.Metabolism,1998,47:663-8.
35 Chen BM,Xia LW,Zhao RQ.Determination of N(G),N(G)-dimethylarginine in human plasma by high-performance liquid chromatography.J Chromatogr B Biomed Sci Appl,1997,692:467-71.
36 Morin C,Zini R,Simon N,et al.Dehydroepiandrosterone and alpha-estradiol limit the functional alterations of rat brain mitochondria submitted to different experimental stresses.Neuroscience,2002,115:415-24.
37 Munujos P,Coll-Canti J,Gonzalez-Sastre F,et al.Assay of succinate dehydrogenase activity by a colorimetric-continuous method using iodonitrotetrazolium chloride as electron acceptor.Anal Biochem,1993,212:506-9.
38 Baracca A,Sgarbi G,Solaini G,et al.Rhodamine 123 as a probe of mitochondrial membrane potential:evaluation of proton flux through F(0)during ATP synthesis.Biochim Biophys Acta,2003,1606:137-46.
39 Scaduto RC Jr, Grotyohann LW. Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives. Biophys J, 1999,76:469-77.
40 Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 1976,72.248-54.
41 Fu YF, Xiong Y, Guo Z. A reduction of endogenous asymmetric dimethylarginine contributes to the effect of captopril on endothelial dysfunction induced by homocysteine in rats. Eur J Pharmacol, 2005,508:167-75.
42 Akao Y, Otsuki Y, Kataoka S, et al. Multiple subcellular localization of bcl-2: detection in nuclear outer membrane, endoplasmic reticulum membrane, and mitochondrial membranes. Cancer Res, 1994,54:2468-71.
43 Kibirige M, Metcalf B, Renuka R, et al. Testing the accelerator hypothesis: the relationship between body mass and age at diagnosis of type 1 diabetes. Diabetes Care, 2003,26:2865-70.
44 Lupi R, Marselli L, Dionisi S, et al. Improved insulin secretory function and reduced chemotactic properties after tissue culture of islets from type 1 diabetic patients. Diabetes Metab Res Rev, 2004,20:246-51.
45 Chavin KD, Yang S, Lin HZ, et al. Obesity induces expression of uncoupling protein-2 in hepatocytes and promotes liver ATP depletion. J Biol Chem, 1999,274:5692-700.
46 Stuart JA, Harper JA, Brindle KM, et al. Physiological levels of mammalian uncoupling protein 2 do not uncouple yeast mitochondria. J Biol Chem, 2001,276:18633-9.
47 Chan CB, De Leo D, Joseph JW, et al. Increased uncoupling protein-2 levels in beta-cells are associated with impaired glucose-stimulated insulin secretion: mechanism of action. Diabetes, 2001,50:1302-10.
48 Nagase I, Yoshida T, Saito M. Up-regulation of uncoupling proteins by beta-adrenergic stimulation in L6 myotubes. FEBS Lett, 2001,494:175-80.
49 Zhang CY, Baffy G, Perret P, et al. Uncoupling protein-2 negatively regulates insulin secretion and is a major link between obesity, beta cell dysfunction, and type 2 diabetes. Cell, 2001,105:745-55.
50 Merial C, Bouloumie A, Trocheris V, et al. Nitric oxide-dependent downregulation of adipocyte UCP-2 expression by tumor necrosis factor-alpha. Am J Physiol Cell Physiol, 2000, 279:C1100-6.
51 Fridlyand LE, Philipson LH. Reactive species and early manifestation of insulin resistance in type 2 diabetes.Diabetes Obes Metab,2006,8:1 36-45.
52 Sydow K,Munzel T.ADMA and oxidative stress.Atheroscler Suppl,2003,4:41-51.
53 Ricquier D,Bouillaud F.The uncoupling protein homologues:UCP1,UCP2,UCP3,StUCP and AtUCP.Biochem J,2000,345 Pt 2:161-79.
54 Mohazzab KM,Kaminski PM,Wolin MS.NADH oxidoreductase is a major source of superoxide anion in bovine coronary artery endothelium.Am J Physiol,1994,266:H2568-72.
55 Jia SJ,Jiang DJ,Hu CP,et al.Lysophosphatidylcholine-induced elevation of asymmetric dimethylarginine level by the NADPH oxidase pathway in endothelial cells.Vascul Pharmacol,2006,44:143-8.
56 Cai H,Harrison DG.Endothelial dysfunction in cardiovascular diseases:the role of oxidant stress.Circ Res,2000,87:840-4.
57 Rasbach KA,Schnellmann RG.Signaling of mitochondrial biogenesis following oxidant injury.J Biol Chem,2007,282:2355-62.
58 Brown GC.Cell biology.NO says yes to mitochondria.Science,2003,299:838-9.
59 Nisoli E,.Clementi E,Paolucci C,et al.Mitochondrial biogenesis in mammals:the role of endogenous nitric oxide.Science,2003,299:896-9.
1 Pinhas-Hamiel O, Zeitler P. Acute and chronic complications of type 2 diabetes mellitus in children and adolescents. Lancet, 2007,369:1823-31.
2 Wild S, Roglic G, Green A, et al. Global prevalence of diabetes: estimates for the year 2000 and projections for 2030. Diabetes Care, 2004,27:1047-53.
3 Lowell BB, Shulman GI. Mitochondrial dysfunction and type 2 diabetes. Science, 2005,307:384-7.
4 St John JC, Amaral A, Bowles E, et al. The analysis of mitochondria and mitochondrial DNA in human embryonic stem cells. Methods Mol Biol, 2006,331:347-74.
5 Szewczyk A, Wojtczak L. Mitochondria as a pharmacological target. Pharmacol Rev, 2002,54:101-27.
6 Cohen BH, Gold DR. Mitochondrial cytopathy in adults: what we know so far. Cleve Clin J Med, 2001,68:625-6, 629-42.
7 Heales SJ, Bolanos JP. Impairment of brain mitochondrial function by reactive nitrogen species: the role of glutathione in dictating susceptibility. Neurochem Int, 2002,40:469-74.
8 Brownlee M. A radical explanation for glucose-induced beta cell dysfunction. J Clin Invest, 2003,112:1788-90.
9 Taniguchi A, Fukushima M, Nakai Y, et al. Factors responsible for the evolution of insulin resistance in Japanese type 2 diabetic patients: Association with atherosclerosis. Diabetes Res Clin Pract, 2007.
10 Roos C, Lidfeldt J, Agardh CD, et al. Insulin resistance and self-rated symptoms of depression in Swedish women with risk factors for diabetes: the Women's Health in the Lund Area study. Metabolism, 2007,56:825-9.
11 Dela F. Other adaptations to training/inactivity in type 2 diabetics and other groups with insulin resistance: emphasis on prevention of CHD. Appl Physiol Nutr Metab, 2007,32:602-6.
12 Petersen KF, Dufour S, Befroy D, et al. Impaired mitochondrial activity in the insulin-resistant offspring of patients with type 2 diabetes. N Engl J Med, 2004,350:664-71.
13 Chiasson JL, Brindisi MC, Rabasa-Lhoret R. The prevention of type 2 diabetes: what is the evidence?. Minerva Endocrinol, 2005,30:179-91.
14 Boden G, Shulman GI. Free fatty acids in obesity and type 2 diabetes: defining their role in the development of insulin resistance and beta-cell dysfunction. Eur J Clin Invest, 2002,32 Suppl 3:14-23.
15 Shulman GI. Cellular mechanisms of insulin resistance. J Clin Invest, 2000,106:171-6.
16 Yu C, Chen Y, Cline GW, et al. Mechanism by which fatty acids inhibit insulin activation of insulin receptor substrate-1 (IRS-1)-associated phosphatidylinositol 3-kinase activity in muscle. J Biol Chem, 2002,277:50230-6.
17 Kraegen EW, Cooney GJ, Ye JM, et al. The role of lipids in the pathogenesis of muscle insulin resistance and beta cell failure in type II diabetes and obesity. Exp Clin Endocrinol Diabetes, 2001,109 Suppl 2:S189-201.
18 RANDLE PJ, GARLAND PB, HALES CN, et al. The glucose fatty-acid cycle. Its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus. Lancet, 1963,1:785-9.
19 Clerk LH, Rattigan S, Clark MG. Lipid infusion impairs physiologic insulin-mediated capillary recruitment and muscle glucose uptake in vivo. Diabetes, 2002,51:1138-45.
20 Boden G, Chen X. Effects of fat on glucose uptake and utilization in patients with non-insulin-dependent diabetes. J Clin Invest, 1995,96:1261-8.
21 Blaak EE, Wolffenbuttel BH, Saris WH, et al. Weight reduction and the impaired plasma-derived free fatty acid oxidation in type 2 diabetic subjects. J Clin Endocrinol Metab, 2001,86:1638-44.
22 Mensink M, Blaak EE, van Baak MA, et al. Plasma free Fatty Acid uptake and oxidation are already diminished in subjects at high risk for developing type 2 diabetes. Diabetes, 2001,50:2548-54.
23 Noguchi T, Kasuga M. [Insulin action and its regulatory mechanisms]. Nippon Rinsho, 2006,64 Suppl 9:121-5.
24 Ferreira-Maldent N, Maillot F, Quilliet L, et al. [Dilated cardiomyopathy, diabetes and deafness related to a mutation of mitochondrial DNA]. Arch Mal Coeur Vaiss, 2007,100:149-52.
25 Petersen KF, Befroy D, Dufour S, et al. Mitochondrial dysfunction in the elderly: possible role in insulin resistance. Science, 2003,300:1140-2.
26 Trifunovic A, Wredenberg A, Falkenberg M, et al. Premature ageing in mice expressing defective mitochondrial DNA polymerase. Nature, 2004,429:417-23.
27 Boudina S, Sena S, O'Neill BT, et al. Reduced mitochondrial oxidative capacity and increased mitochondrial uncoupling impair myocardial energetics in obesity. Circulation, increased mitochondrial uncoupling impair myocardial energetics in obesity.Circulation,2005,112:2686-95.
28 Kelley DE,He J,Menshikova EV,et al.Dysfunction of mitochondria in human skeletal muscle in type 2 diabetes.Diabetes,2002,51:2944-50.
29 Short KR,Nair KS,Stump CS.Impaired mitochondrial activity and insulin-resistant offspring of patients with type 2 diabetes.N Engl J Med,2004,350:2419-21;author reply 2419-21.
30 Wu Z,Puigserver P,Andersson U,et al.Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1.Cell,1999,98:115-24.
31 Mootha VK,Lindgren CM,Eriksson KF,et al.PGC-lalpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes.Nat Genet,2003,34:267-73.
32 Patti ME,Butte AJ,Crunkhorn S,et al.Coordinated reduction of genes of oxidative metabolism in humans with insulin resistance and diabetes:Potential role of PGC1 and NRF1.Proc Natl Acad Sci U S A,2003,100:8466-71.
33 Irrcher I,Adhihetty PJ,Sheehan T,et al.PPARgamma coactivator-lalpha expression during thyroid hormone- and contractile activity-induced mitochondrial adaptations.Am J Physiol Cell Physiol,2003,284:C 1669-77.
34 Chan CB,De Leo D,Joseph JW,et al.Increased uncoupling protein-2 levels in beta-cells are associated with impaired glucose-stimulated insulin secretion:mechanism of action.Diabetes,2001,50:1302-10.
35 Zhang CY,Baffy G,Perret P,et al.Uncoupling protein-2 negatively regulates insulin secretion and is a major link between obesity,beta cell dysfunction,and type 2diabetes.Cell,2001,105:745-55.
36 Kassis N,Bernard C,Pusterla A,et al.Correlation between pancreatic islet uncoupling protein-2(UCP2)mRNA concentration and insulin status in rats.Int J Exp Diabetes Res,2000,1:185-93.
37 Brown JE,Thomas S,Digby JE,et al.Glucose induces and leptin decreases expression of uncoupling protein-2 mRNA in human islets.FEBS Lett,2002,513:189-92.
38 Krempler F,Esterbauer H,Weitgasser R,et al.A functional polymorphism in the promoter of UCP2 enhances obesity risk but reduces type 2 diabetes risk in obese middle-aged humans.Diabetes,2002,51:3331-5.
39 Sesti G,Cardellini M,Marini MA,et al.A common polymorphism in the promoter of UCP2 contributes to the variation in insulin secretion in glucose-tolerant subjects. Diabetes, 2003,52:1280-3
40 Sasahara M, Nishi M, Kawashima H, et al. Uncoupling protein 2 promoter polymorphism -866G/A affects its expression in beta-cells and modulates clinical profiles of Japanese type 2 diabetic patients. Diabetes, 2004,53:482-5.
41 Mattiasson G, Sullivan PG. The emerging functions of UCP2 in health, disease, and therapeutics. Antioxid Redox Signal, 2006,8:1-38.
42 Kelley DE, Mintun MA, Watkins SC, et al. The effect of non-insulin-dependent diabetes mellitus and obesity on glucose transport and phosphorylation in skeletal muscle. J Clin Invest, 1996,97:2705-13.
43 Szendroedi J, Schmid AI, Chmelik M, et al. Muscle mitochondrial ATP synthesis and glucose transport/phosphorylation in type 2 diabetes. PLoS Med, 2007,4:e154.
44 Kashemsant N, Chan CB. Impact of uncoupling protein-2 overexpression on proinsulin processing. J Mol Endocrinol, 2006,37:517-26.
45 Echtay KS, Murphy MP, Smith RA, et al. Superoxide activates mitochondrial uncoupling protein 2 from the matrix side. Studies using targeted antioxidants. J Biol Chem, 2002,277:47129-35.
46 Krauss S, Zhang CY, Scorrano L, et al. Superoxide-mediated activation of uncoupling protein 2 causes pancreatic beta cell dysfunction. J Clin Invest, 2003,112:1831-42.
47 Langin D. Diabetes, insulin secretion, and the pancreatic beta-cell mitochondrion. N Engl J Med, 2001,345:1772-4.
48 Bindokas VP, Kuznetsov A, Sreenan S, et al. Visualizing superoxide production in normal and diabetic rat islets of Langerhans. J Biol Chem, 2003,278:9796-801.
49 Brownlee M. Biochemistry and molecular cell biology of diabetic complications. Nature, 2001,414:813-20.
50 El Midaoui A, de Champlain J. Prevention of hypertension, insulin resistance, and oxidative stress by alpha-lipoic acid. Hypertension, 2002,39:303-7.
51 Du Y, Miller CM, Kern TS. Hyperglycemia increases mitochondrial superoxide in retina and retinal cells. Free Radic Biol Med, 2003,35:1491-9.
52 Korshunov SS, Skulachev VP, Starkov AA. High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria. FEBS Lett, 1997,416:15-8.
53 Du XL, Edelstein D, Dimmeler S, et al. Hyperglycemia inhibits endothelial nitric oxide synthase activity by posttranslational modification at the Akt site. J Clin Invest, 2001,108 1341-8
54 Victor VM, Rocha M. Targeting antioxidants to mitochondria a potential new therapeutic strategy for cardiovascular diseases. Curr Pharm Des, 2007,13:845-63.
55 Nishikawa T, Edelstein D, Du XL, et al. Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage. Nature, 2000,404:787-90.
56 Du XL, Edelstein D, Rossetti L, et al. Hyperglycemia-induced mitochondrial superoxide overproduction activates the hexosamine pathway and induces piasminogen activator inhibitor-1 expression by increasing Sp1 glycosylation. Proc Natl Acad Sci U S A, 2000,97:12222-6.
57 Du X, Matsumura T, Edelstein D, et al. Inhibition of GAPDH activity by poly(ADP-ribose) polymerase activates three major pathways of hyperglycemic damage in endothelial cells. J Clin Invest, 2003,112:1049-57.