| Chapter 1 Effects of Asymmetric Dimethylarginine on Erythrocyte- Deformability in Diabetic RatsBACKGROUNDNormal deformability of erythrocytes is essential in maintaining microcirculatory perfusion and the transportation of oxygen and nutrients. It had been documented that nitric oxide(NO) participated in modulation of erythrocyte deformability,and that endogenous nitric oxide synthase (NOS) inhibitors asymmetric dimethylarginine(ADMA),which inhibited the production of NO,increased in diabetic animals and patients.ADMA, besides inhibiting NOS,can induce oxidant stress.Recent studies showed that erythrocyte deformability was decreased concomitantly with increased plasma level of ADMA in patients with hypertension.Since ADMA was also elevated in type 1 diabetes mellitus,we examined the effect of ADMA on erythrocyte deformability in type 1 diabetic rats.METHODSA single intraperitoneal injection of streptozotocin(STZ,65mg/kg) in male Sprague-Dawley rats was carried out to induce diabetes and normal erythrocytes were incubated with asymmetric dimethylarginine or aortic rings of diabetic rats in the presence of L-arginine or vitamin E. The effects of ADMA on erythrocytes deformability,and contents of NO, reactive oxygen species(ROS) and malondialdehyde(MDA) were examined.RESULTS(1) Erythrocyte deformability was significantly decreased in diabetic rats.The levels of asymmetric dimethylarginine in plasma and erythrocytes of diabetic rats were elevated significantly from 2-week duration to 8-week duration.Nitric oxide in erythrocytes was decreased at 8-week duration while plasma nitric oxide remained unchanged all along. The content of MDA in erythrocytes of diabetic rats was increased.(2) After incubation of erythrocytes with asymmetric dimethylarginine(10-6 M) for 30 minutes,erythrocyte deformability and nitric oxide level in erythrocytes were decreased markedly.ROS and MDA production in erythrocytes were increased in the presence of asymmetric dimethylarginine.Both L-arginine and vitamin E reversed the effects of asymmetric dimethylarginine.(3) After incubation of erythrocytes with aortic rings of diabetic rats,erythrocyte deformability was decreased,which was attenuated by L-arginine.CONCLUSIONReduction of erythrocyte deformability in diabetic rats was associated with elevation of asymmetric dimethylarginine,which increased oxidant and impaired nitric oxide synthesis.Chapter 2 Insulin resistance in diabetes: role of DDAH/ADMA system in adipocytes.BACKGROUNDInsulin resistance is the important characteristics of type 2 diabetes, polycystic ovary syndrome and other diseases.It is always characterized with decreased insulin sensitivity in liver,muscle and adipose tissue, resulting in less biological effects of insulin than normal.Recent studies suggest that except for storage of fat and energy,adipose tissue also acts as an endocrine organ.Adipose tissue produces many kinds of cytokines, which plays an important role in regulation of adipose function.Recent study reveals that adipocytes can also secret ADMA.Therefore,the present study is mainly design to explore whether DDAH/ADMA system in adipocytes is involved in insulin-resistance in type 2 diabetic rats.METHODS(1) In insulin-resistant diabetic rat model,the levels of dimethylarginine-dimethylamine hydrolase(DDAH) and ADMA in adipose tissue were examined.Correlation analysis was also performed to explore the relationship between DDAH/ADMA system and insulin sensitivity.(2) After cultured 3T3-L1 adipocytes were treated with higher concentration of glucose(25 mM),activity and expression of DDAH, and levels of ADMA,insulin receptor substrate-1(IRS-1) and glucose transportor-4(GLUT4) were examined.The influence of human-DDAH2 gene(hDDAH) over-expression on the effects of high concentration of glucose was observed.RESULTS(1) In diabetic rats,levels of blood glucose and AUC(area of under curve) of OGTT were significantly increased,while plasma insulin decreased.HOMA-IR of diabetic rat was higher than that of control rats.(2) Compared with control rats,the expression of DDAH2,the activity of DDAH,and contents of NOS and NO were decreased significantly in adipose tissue of diabetic rats,while ADMA contents increased.The expression of IRS-1 and GLUT4 mRNA were down-regulated in diabetic rats.(3) DDAH and NOS activity,and NO contents in adipose tissue was negatively correlated to blood glucose,area under curve(AUC) of OGTT and HOMA resistance index,respectively,while positively correlated to plasma insulin.ADMA was positively correlated to blood glucose,area under curve(AUC) of OGTT and HOMA resistance index,respectively. (4)Treatment with high glucose for 72 h decreased the expression of DDAH2 mRNA in 3T3-L1 adipocytes.(5) Over-expression of hDDAH2 increased DDAH activity in 3T3-L1 adipocytes,concomitantly with an increase in ADMA contents. Over-expression of hDDAH2 also reversed inhibited IRS-1and GLUT4 mRNA expression by high glucose.CONCLUSIONImpairment of insulin signal transduction in type 2 diabetes was associated with decreased DDAH activity as well as increased ADMA contents in adipocytes.ADMA might be a new adipocytokine participating in the development of insulin resistance.Chapter 3 Asymmetric Dimethylarginine induces insulin resistance in adipocytesBACKGROUNDAsymmetric dimethylarginine(ADMA),the inhibitor of nitric oxide synthase(NOS),has been reported to be associated with glucose metabolism and insulin sensitivity.Our previous study also demonstrates that DDAH/ADMA system participates in insulin signal transduction. Moreover,mechanisms responsible for the effect of DDAH/ADMA system on insulin signal transduction remain unknown.It has been reported that oxidative stress or toll-like receptor 4 (TLR4)-mediated inflammation is involved in the development of adipocyte dysfunction,and proximal events in TLR4 signaling has been shown to be oxidant-dependent.Recently,it has been reported that ADMA participates in oxidative stress or inflammatory reactions.In the present study,therefore,we examined the effects of exogenous ADMA on glucose transport in adipocytes its mechanisms.METHODSAfter cultured 3T3-L1 adipocytes were treated with ADMA(1,3,10μM),glucose uptake and the expression of insulin receptor substrate-1 (IRS-1) and glucose transporter-4(GLUT4) were examined.The levels of nitric oxide(NO),reactive oxygen species(ROS) and tumor necrosis factor alpha(TNF-α),and the expression of toll-like receptor 4(TLR4) were also measured.RESULTS(1) ADMA significantly impaired basal or insulin-stimulated 2-deoxy-[3H]glucose uptake,and down-regulated the expression of IRS-1 and GLUT4.(2) Phosphorylated protein of IRS-1 and translocation of GLUT4 with insulin-stimulation were inhibited by ADMA.(3) The level of NO was decreased,while contents of ROS and TNF-α,and expression of TLR4 were increasedin the presence of ADMA.(4) Vitamin E reduced the inhibitory effects of ADMA on glucose transport,on the levels of NO and ROS,and on the expression of TLR4. Moreover,vitamin E decreased ADMA contents and increased dimethylarginine dimethylaminohydrolase(DDAH) activity.L-arginine increased NO level,but failed to reduce the action and content of ADMA.CONCLUSIONImpairment of both basal and insulin-stimulated glucose transport by ADMA may relate to activation of the ROS / TLR4 pathway in adipocytes.
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