Role Of Asymmetric Dimethylarginine In The Development Of Skeletal Muscle Insulin Resistance In Hyperlipemic Rabbits And Its Potential Mechanisms

BACKGROUND Insulin resistance is the primary cause and pathological basis for type 2 diabetes, and is also the common risk factor of cardiovascular diseases such as hypertension and atherosclerosis. Impairment of insulin-stimulated glucose uptake and utilization in skeletal muscle is the main characteristics of insulin resistance. Accumulating evidence showed that activation of phosphatidylinositol 3-kinase (PI3K) pathway plays a central role in insulin-stimulated glucose transport in skeletal muscle. After binding with insulin receptor (IR) on the surface of skeletal muscle cells, insulin causes tyrosine phosphorylation of insulin receptor substrate (IRS) proteins, which then associate with PI3K through the p85 regulatory domain, leading to increased PI3K activity. This then leads to activation of Akt2, which ultimately can result in insulin-stimulated glucose transporter-4 (Glut-4) translocation and increased glucose transport. Recent studies have shown that activation of PI3K could also cause nitric oxide (NO) release and NO is directly involved in insulin's regulatory effects on glucose uptake by mediating vasodilatation in skeletal muscle. Therefore, NO has been considered as a new key effector molecule in maintenance of metabolic homeostasis. Numerous enviromental factors including high-fat diet block the expression of key molecules in insulin signalling pathway, leading to insulin resistance in skeletal muscle. The elevation of endogenous nitric oxide synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA) is frequently observed in high-fat associated insulin resistant state. Endogenous ADMA was degraded by dimethylarginine dimethylaminohydrolase (DDAH). Since NO plays an important role in the regulation of glucose metabolism, and ADMA is a critical determinant of NO contents, we presumed that ADMA might play a role in the development of insulin resistance via affecting NO levels. Therefore, the present study was designed to examine the alterations of DDAH/ADMA/NOS/NO pathway and insulin PI3K signal transduction in the skeletal muscle from high-fat fed insulin resistant rabbits, and to explore the effects of ADMA on insulin-stimulated glucose uptake and IR,IRS-1,IRS-2,PI3K p85 subunit, Akt2 and Glut4 expression in mouse skeletal muscle cell line C2C12. In additon, we also observed whether captopril, an angiotensin-converting enzyme inhibitor, could enhance DDAH activity, decrease endogenous ADMA levels, restore NOS activity and NO synthesis, and reverse the impairment of PI3K transduction in skeletal muscle from high-fat fed rabbits in addition to protecting against endothelial dysfuntion in hyperlipemia, and improve insulin resistance and the impairment of PI3K signal transduction in C2C12 cells induced by ADMA. These results may provide novel experimental evidence for the role of ADMA in the development of insulin resistance and its potential mechanisms, and further provide new insight into the clinical prevention and treatment of insulin resistance.METHODS①Rabbits were given a high-fat diet to induce insulin resistance and some animals were treated with captopril(8 mg/kg/d) for 12 weeks. Parameters including body weight, serum glucose, insulin levels, lipid profiles and morphological changes of liver were determined to assess insulin sensitivity in a comprehensive way. Isometric tension recordings were used to measure endothelium-dependent relaxation in response to acetylcholine. ADMA was analysed by high performance liquid chromatography. DDAH activity, NOS activity and NO contents in skeletal muscle were also determined to reflect the alterations of DDAH/NOS/NO pathway in animals. Reverse transcription- polymerase chain reaction (RT-PCR) was applied to detect the expression of IR,IRS-1,IRS-2,PI3K p85 subunit, Akt2, Glut4 in skeletal muscle. In addition, serum malondialdehyde levels were assayed to reflect the lipid peroxidation in vivo.②C2C12 cells were incubated with exogenous ADMA, and the amount of [~3H]-2-deoxy-glucose uptaked by C2C12 cells was determined by liquid scintillation as an indicator of cellular insulin sensetivity. RT-PCR was used to detect gene expression of IR,IRS-1,IRS-2,PI3K p85 subunit, Akt2 and Glut4. Malondialdehyde contents in the media were measured with thiobarbituric acid method.RESULTS①High-fat fed rabbits developed obesity, hyperglycemia, hyperinsulinimia, disorders of lipid metabolism and hepatic fatty degeneration. The insulin sensitivity index was significantly decreased, indicating that insulin resistant animal model was successfully established. Endothelium-dependent relaxation of aortic rings from insulin resistant rabbis was significantly impaired (56±3 % vs 94±2 %, P<0.01 vs Con); Serum ADMA levels were markedly elevated (2.24±0.12 vs 1.22±0.12μmol/L, P<0.01 vs Con), and muscular DDAH activity was significantly inhibited (0.19±0.01 vs 0.30±0.02 U/g pro, P<0.01 vs Con). These results were associated with a reduction of muscular NOS activity, NO contents, and a downregulation of Glut4 mRNA expression. In contrast, serum malondialdehyde levels were significantly elevated in high-fat fed rabbits. However, high-fat fed had no significant effects on IR,IRS-1,IRS-2,PI3K p85 subunit and Akt2 expression. Captopril treatment not only restored the changes of the above indexes but also improved insulin sensitivity. But captopril adminstration had no significant effects on body weight and lipid profile, neither on the mRNA expressions of IR,IRS-1,IRS-2,PI3K p85 subunit and Akt2.(2) Exogenous ADMA decreased insulin-stimulated [~3H]-2-deoxy-glucose uptake by C2C12 cell in a dose- and time-dependent manner, indicating that insulin sensitivity was reduced in C2C12 myotubes after exposure to ADMA. Incubation of cells with 30μmol/L ADMA for 48h markedly inhibited Glut4 rather than IR,IRS-1,IRS-2,PI3K p85 subunit, Akt2 gene espression. In addition, malondialdehyde levels were significantly elevated by ADMA incubation. Preincubation with captopril reversed these effects of ADMA on C2C12 myotubes, which was similar with PDTC, an efficient antioxidant.CONCLUSIONS①It was found in animal experiments that high fat induced insulin resistance was closely related to an elevation of serum ADMA, suppression of DDAH/NOS/NO pathway, and downregulation of Glut4 gene expression in skeletal muscle. Captopril treatment decreased endogenous ADMA contents, restored muscular NOS activity and NO synthesis, and Glut4 mRNA expression, which may be the underlying mechanisms responsible for the favorable effects of captopril on insulin sensitivity.②In cultured myotubes, ADMA decrease insulin sensitivity via enhancing oxidative stress and down-regulating Glut4 expression. Captopril could prevent the adverse effects of ADMA on cellular insulin sensitivity.