The Role Of Asymmetric Dimethylarginine In Hepatic Mitochondrial Dysfunction Of Diabetic Rats

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.