Atorvastain Modulates DDAH1/ADMA System Via SREBP1Pathway In Insulin Resistant Condition

Chapter1. Atorvastatin modulates DDAH1/ADMA system in high-fat diet-induced insulin resistant rats with endothelial dysfunctionObjective:Endothelial dysfunction, a main risk factor of cardiovascular diseases, can be attributed to insulin resistance. Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), plays an important role in endothelial function and has been considered as a biomarker for major cardiovascular events and mortality in cohorts with high, intermediate, and low overall cardiovascular risks. Dimethylarginine dimethyl-aminohydrolase1(DDAH1) is a metabolic enzyme for asymmetric dimethylarginine (ADMA), both of which are closely related to endothelial function. Atorvastatin has been widely used in cardiovascular disease to protect endothelial function. We established insulin resistant rats models and observed the effects of atorvastatin on DDAH1/ADMA system.Methods:Eight-week-old SD rats (weighed200-250g) were randomly divided into two groups:(1) the control group (CON; n=10), in which rats were fed with standard rodent chow and water adlibitum (protein,20kcal%; carbohydrate,70kcal%; and lipid,10kcal%) and (2) the high-fat diet group (HFD; n=20), in which rats were fed with fat-rich chow and water adlibitum (protein,20kcal%; carbohydrate,35kcal%; and lipid,45kcal%, predominantly in the form of lard). At the end of the8th week, fasting plasma glucose and insulin levels were measured, and insulin sensitivity was evaluated by calculating the Homeostatic Model Assessment-Insulin Resistance (HOMA-IR) index as [(fasting insulin, mU/ml)×(fasting glucose, mmol/L)/22.5]. Studies have previously demonstrated that high-fat diet induced insulin resistance in rats. Next, the HFD group was further divided into two group. The first group (IR+A; n=10) received atorvastatin (30mg/kg/day; Pfizer Pharmaceuticals) and the second group (IR; n=10) was kept on vehicle (water) for additional8weeks. Body weights were measured weekly. Fasting glucose and insulin levels were measured at the beginning of the8th and16th week. Finally, the aorta and plasma were collected and stored at-80℃. At the end of study, protein and mRNA were examined by using Western-blot and Real-time PCR, Plasma ADMA concentrations were measured by high-performance liquid chromatography.Results:1. Higher levels of plasma triglycerides and C reactive protein (CRP) were observed in insulin resistant rats than those in the control (1.45±0.41vs.0.9±0.24mmol/L, P<0.05;0.3±0.1vs.0.12±0.1mg/L, P<0.01; respectively). Significantly, atorvastatin treatment reduced both plasma triglycerides and CRP levels in insulin resistant rats (0.82±0.3mmol/L and0.03±0.01mg/L, respectively; P<0.01).2. High-fat diet-fed rats showed the significant higher HOMA-IR than the control group (3.06±0.6vs.1.89±0.3, P<0.05), and such high insulin sensitivity in insulin resistance rats was reduced by atorvastatin treatment (3.06±0.6vs.2.38±0.5, P<0.05).3. Plasma ADMA concentrations were found significantly increased in insulin resistance rats, compared to the control (1.1±0.24vs.0.52±0.1μmol/L, P<0.01), whereas aortic DDAH activity was reduced (0.05±0.01vs.0.12±0.01μ/g protein, P<0.01). Further atorvastatin treatment of insulin resistance rats not only significantly decreased plasma ADMA levels (0.87±0.22vs.1.1±0.24μmol/L, P<0.05) but also enhanced aortic DDAH activity by18%(0.07±0.012vs.0.05±0.01μ/g protein, P<0.05).4. A significant negative correlation between HOMA-IR and aortic DDAH activity (r=-0.795, P<0.01) was observed, which in turn indicates a positive correlation between insulin sensitivity and aortic DDAH activity.5. Insulin resistant rats showed decreased plasma NO levels and NOS activity, compared to the control group (25.6±7.4vs.62.4±4.9μmol/L,19±3.2vs.35±4.8μ/ml, respectively; P<0.01). Significantly, atorvastatin treatment of insulin resistance rats was able to increase the plasma NO levels (43.53±8.2vs.25.6±7.4μmol/L, P<0.01) and NOS activity (26±3.5vs.19±3.2μ/ml, P<0.05).6.Insulin resistance in high-fat diet-fed rats significantly attenuated ACh-induced endothelium-dependent relaxation; in contrast, atorvastatin treatment preserved the control-level relaxation7. The mRNA and protein expression of SREBP1and DDAH1in thoracic aorta of insulin resistant rats were significantly lower than those in the control; however, all levels were restored by further atorvastatin treatment.Conclusion:High-fat diet-induced insulin resistance was able to not only downregulate aortic DDAH1expression and DDAH activity but also increase plasma ADMA concentrations in rats. Furthermore, atorvastatin treatment increased the expression of both SREBP1and DDAH1in thoracic aorta and decreased plasma ADMA levels in insulin resistant rats. These results suggest that atorvastatin may protect endothelial function by modulating the DDAH1/ADMA system, specifically, by restoring DDAH activity in insulin resistant rats. Chapter2Atorvastain modulates DDAH1/ADMA system via SREBP1pathway in insulin resistant HUVECsObjective:Dimethylarginine dimethyl-aminohydrolase1(DDAH1)/Asymmetric dimethylarginine (ADMA) system is closely related to endothelial function. Atorvastatin performed as an endothelium-protective drug, and in vivo study we have proved that atorvastatin modulated DDAH1/ADMA system in insulin resistant rats. However, the possible mechanism is not clear. Sterol regulatory element binding protein-1(SREBP1), a transcription factor regulating the expression of genes involving in lipid homeostasis and glucose metabolism, was reported to be regulated by Atorvastatin in the progress of lipid lowering. Considering the promoter of DDAH1contains SREBP1binding sites, we make a hypothesis that atorvastatin can modulate DDAH1/ADMA system via SREBP1pathway. We aimed to determine the possible mechanism of atorvastatin on DDAH1/ADMA in Human umbilical vein endothelial cells (HUVECs)Methods:Human umbilical vein endothelial cells (HUVECs) were treated in RPMI1640medium supplemented with1%FBS and10,50,100,500,1000nmol/L insulin for12,24,36,48hours respectively. The glucose consumption were used to determine the insulin sensitivity. After the establishment of insulin resistant cell model, siRNA for silencing SREBP-1were transfected into HUVEC cells at a final concentration of50nM according to the manufacturer’s protocol. a master mix of Lipofectamine2000was diluted with1ml of OPTI-MEM (Invitrogen) and incubated for5min. Lipofectamine2000dilution was added to the DNA/siRNA dilution, incubated for20min and added drop-wise to the cells. Five hours after transfection, the media was changed and the cells were allowed to recover overnight The gene and protein of SREBPlwere tested to evaluate the transfect efficiency. The total content of nitrite and nitrate were measured to reflect NO level. ADMA concentrations in medium were measured by high-performance liquid chromatography (HPLC) using precolumn derivatization with ophthaldialdehydeas. The cell DDAH activity was measured by determining L-citrulline formation. Western immunoblotting and Realtime PCR were carried out to determine the protein and mRNA expressed in endothelial cells.Results:1. Endothelial cells treated with100nmol/L insulin for24hours showed the least glucose consumption, which indicate an insulin resistant cell model.2. The medium ADMA concentrations were significantly increased in insulin resistant group (IR) compared with control group (CON), whereas both NO concentration DDAH activity were reduced in insulin resistant groups. Treatment with atorvastatin significantly decreased plasma ADMA level and enhanced DDAH activity and NO production in HUVECs.3. SREBP1and DDAH1mRNA expression were decreased in insulin resistant HUVECs. In consistent with gene expression, SREBP1and DDAH1protein expression were also reduced significantly. With atorvastatin treatment, both mRNA and protein expression of SREBP1and DDAH1were increased in HUVECs.4. SREBP-1siRNA successfully knocked down SREBP-1mRNA and protein levels in HUVECs in insulin-resistant condition. Whereas, the scramble siRNA had no effect on the expression of SREBP-1. Treatment with siRNA targeting SREBP-1caused a73%decrease in SREBP-1mRNA and a79%decrease in protein in comparison with scramble siRNA transduced cells in insulin-resistant condition. And SREBP-1knockdown is associated with a75%decrease in DDAH1mRNA and a69%decrease in protein expression in high glucose cultured HUVECs. Moreover, SREBP1knockdown decreased the expression of DDAH1in HUVECs treated with atorvastatin significantly.5. SREBP-1knockdown resulted a64%decrease in DDAH activity compared with scramble siRNA transduced cells, accompanying a2.9-fold increase in ADMA concentration. atorvastatin treatment induced an additional increase in DDAH activity in SREBP-1knockdown cells, but there is no statistical significance. Levels of NO indicated the activity of NOS isoforms. SREBP-1knockdown was associated with a62%decrease in NO levels. Moreover, SREBP1knockdown led to a significant decrease of DDAH activity and NO level in HUVECs treated with atorvastatin, accompanying an increase of ADMA level.Conclusion:1. Atorvastatin benefits endothelial function by modulating DDAH1/ADMA/NO axis in insulin resistant HUVECs.2. SREBP1acts as a mediator in the regulation of DDAH1/ADMA system by atorvastatin. Further efforts are required to investigate the concrete mechanism of SREBP1in modulating DDAH1expression and to validate the effects of SREBPs on DDAH1.