The Effect Of SREBP/DDAH/ADMA Pathway On Vascular Endothelial Injury In Hyperinsulinemia

Chapter1The effect of hyperinsulinemia on plasma asymmetric dimethylarginine in patients with coronary heart diseaseObjectives:Hyperinsulinemia occurs in the early stage of type2diabetes mellitus (T2DM) and the period using insulin as therapy. Hyperinsulinemia, promoting atherosclerosis, is an independent risk factor for coronary heart disease. Since endothelial dysfunction is the initiating factor of atherosclerosis, endothelial-derived NO reduction in endothelial represents endothelial damage. Endogenous NOS inhibitor, asymmetric dimethylarginine (ADMA), decreases NO synthesis, as a predictor of cardiovascular death. The present study aims to investigate the impact of hyperinsulinemia on human plasma ADMA, and want to provide the basis for the damage of ADMA on endothelium by hyperinsulinemia.Methods:89people from September2009to May2010in Department of Cardiology, and Medical Examination Center, Xiangya Hospital, Central South University were enrolled in the present study. Cases in3different groups are as:(1) Normal Control group (Con):N=30;(2) Coronary Artery Disease group (CAD): N=30;(3) Coronary Artery Disease combined with Hyperinsulinemia group (CAD+Hins):N=29. The liver function, plasma fasting blood glucose (FBG), lipids (TC, TG, LDL-C, HDL-C), renal function (UA, BUN, Cr), high-sensitivity C-reactive protein (hs-CRP) were all checked after collecting5ml after10h overnight fasting blood samples, by automatic biochemical analyzer. Fasting insulin (Fins) and2h postprandial insulin (2h-Pins) were measured by fully automatic markers of immune chemi-luminescence analyzer. Meanwhile, the age, gender, height, weight and the waistline were recorded, the blood pressure was checked, and the body mass index (BMI) was calculated for all the subjects.Results:1. Gender, age, blood pressure, liver function have no significant difference between CAD+Hins group, CAD group, and Con group.2. The BMI of the CAD+Hins group, and FPG, Fins,2h-Pins, HOMA-1R were significantly higher than the CAD group and Con group (P<0.05). And indicators above in CAD group was significantly higher than Con group (P<0.05).3. TC has no significant difference between the groups. TG, HDL-C, and UA, Cr, BUN were significantly higher in CAD+Hins, and CAD group than that of Con group (P<0.05). LDL-C of CAD group was significantly higher than Con group (P<0.05), and UA of CAD+Hins group was significantly higher than CAD group (P<0.05).4. ADMA, hs-CRP levels in the CAD+Hins group were significantly higher than both of CAD group and Con group (P<0.05); ADMA in CAD group was significantly higher than in Con group (P<0.05).5. No correlation between plasma ADMA and other biochemical markers.Conclusions:1. Hyperinsulinemia enhanced the metabolic disorders on sugar, lipid, and uric acid metabolism in patients with coronary artery disease.2. Hyperinsulinemia further increased the plasma ADMA levels in patients with coronary artery disease, which means the aggravation of endothelial dysfunction.3. Plasma hs-CRP levels was higher in coronary artery disease patients combined with hyperinsulinemia, suggesting that hyperinsulinemia has relationship with chronic inflammatory response. Chapter2Changes of plasma ADMA/NO and aortic SREBPs, DDAH in hyperinsulinemia rats, and their relationship with aortic intimal injuryObjectives:Clinical study in Chapter1has found that plasma asymmetric dimethylarginin (ADMA) level was significantly higher in coronary artery disease patients accompany by hyperinsulinemia than simple coronary artery disease and healthy controls, suggesting that high insulin concentration might injure endothelial function. Dimethylarginine dimethylaminohydrolate (DDAH), a key enzyme regulating ADMA concentrations, contains sterol regulatory element (SRE) on it’s promoter region, which allows sterol regulatory element binding proteins (SREBPs) combine with it and regulate DDAH expression. Since high insulin concentration promotes SREBPs expression, investigation of hyperinsulinemia effect on expression of DDAH through SREBPs regulation pathway might help us clarify the mechanism of high insulin concentration on endothelial injure. This study is to set up hyperinsulinemia SD rat model firstly, and then clarify directly the injury and the possible mechanism on aortic intima by hyperinsulinemia.Methods:Establish the insulin-treated animals and control group: hyperinsulinemia SD rat model group (Hins):animals underwent twice daily sc injection of insulin (N=7), and control SD rats group (Con)(N=7). Measurement of body weight before killing the rats, tail vein blood for checking fasting blood glucose (FBG), and taking fasting blood samples by cardiac puncture. The plasma lipids (TC, TG, LDL-C, HDL-C), high-sensitivity C-reactive protein (hs-CRP) were all checked by automatic biochemical analyzer. Fasting insulin (Fins) was measured by ELISA method, and then calculated the HOMA-IR. Colorimetry method for plasma NO concentration, and HPLC method for ADMA concentration were all done. Thoracotomy immediately after the rats were sacrificed, and ascending aorta organizations was isolated and stored in liquid nitrogen rapidly,4%formaldehyde fixed meanwhile. Test the proteins expression of SREBP-1, SREBP-2, DDAH1, DDAH2by Western-Blotting and the corresponding gene expression by RT-PCR.Results:1. The FBG in Hins group, and the insulin, HOMA-IR, and triglyceride (TG) were significantly higher than Con rats (P<0.05), which suggests the successful hyperinsulinemia SD rat model.2. The body weight in Hins group was significantly higher than Con group (P<0.05).3. There is no significant difference in TC, LDL-C, and HDL-C between two groups. The ADMA and hs-CRP levels were both significantly higher than Con group (P<0.05), while the NO concentration was significantly lower than Con group (P<0.05).4. Compared with Con rats, SREBP-1and SREBP-2protein and gene expression in Hins rats aortic tissue were significantly increased, while DDAH1, DDAH2results contrasted with the results of SREBPs (P<0.05).5. Aortic morphological changes:aortic intimal looks smooth and clarity, and the cells arranged in neat rows in Con group rat. While the aortic intima looks irregular and the cells looks disorder in the Hins group rats.Conclusions:1. Hyperinsulinemia SD rat model can be created by continuous subcutaneous insulin injection, which provides a viable animal model for studying the effect of hyperinsulinemia.2. Plasma ADMA concentration increased, while NO concentration decreased in hyperinsulinemia SD rat, which indicates endothelial dysfunction.3. The aortic intima morphological changes in hyperinsulinemia SD rat due to endothelial injury.4. Proteins and genes expressions of SREBP-I and2in aortic tissue of hyperinsulinemia SD rat increased, while DDAH1and2decreased. It might be the new explanation of the mechanism on the vascular endothelial injury by hyperinsulinemia. Chapter3The affects and mechanisms of high insulin on the expressions of DDAH1, DDAH2gene and protein in HUVECObjective:Hyperinsulinemia is an important risk factor of cardiology incidents. We have found that high insulin suppresses the expressions of DDAH gene and protein from former investigations in vivo. Based on the former studies, in this chapter, we move to a forward step to investigate the affect of high concentration insulin on DDAH1and DDAH2expression in human umbilical vein endothelial cells (HUVEC). Also, we will apply the siRNA transfection technology to silent SREBP-1and SREBP-2expression, and then clarify the mechanism of endothelial dysfunction by high hyperinsulinemia.Methods:1. Using different concentrations of insulin as treatments on HUVEC. The HUVEC were divided into two groups as treated with24-hour and48-hour, the glucose uptake rate was measured, and the effect of insulin concentration, different treat time on the cellular glucose uptake ability were analyzed.2. Human umbilical vein endothelial cells were divided into two groups after successful completion of the siRNA transfection preliminary experiments:one is for siRNA-SREBP1and the other is for siRNASREBP2. The details of the groups are described as:Experiment for siRNA-SREBP-1:(1) Al:normal culture HUVEC for48h;(2) B1:HUVEC cultured in6×10-8mol/L insulin for48h;(3) Cl:siRNA-NC+Insulin group:HUVEC transfected with siRNA-NC, then cultured in6×10-8mol/L insulin for48h;(4) D1: siRNA-SREBP-1group:HUVEC transfected with siRNA-SREBP-1, cultured in normal condition for48h;(5) El:siRNA-SREBP-1+Insulin group:HUVEC transfected with siRNA-SREBP-1, cultured in6×10-8mol/L insulin for48h. And the experiment for siRNA-SREBP-2:(6) A2:normal culture HUVEC for48h;(7) B2: HUVEC cultured in6×10-8mol/L insulin for48h;(8) C2:siRNA-NC+Insulin group: HUVEC transfected with siRNA-NC, then cultured in6×10-8mol/L insulin for48h;(9) D2:siRNA-SREBP-2group:HUVEC transfected with siRNA-SREBP-2, cultured in normal condition for48h;(10) E2:siRNA-SREBP-2+Insulin group: HUVEC transfected with siRNA-SREBP-2, then cultured in6×0-8mol/L insulin for48h. The mRNA and protein of SREBP-1, SREBP-2, DDAH1and DDAH2of every group were extracted for detection of gene expression with using Real-Time PCR technology, and the protein expression of SREBP-1, SREBP-2, DDAH1and DDAH2were detected by Western-Blot.Results:1. Low concentration insulin improved the glucose uptake rate of HUVEC, and the glucose uptake rate was increased gradually with the increasing of insulin concentration. But when insulin concentration is over one point, the glucose uptake rate was declined instead.2.6×10-8mol/L insulin, with48h treatment significantly induced the expression of SREBP-1and SREBP-2genes and protein in HUVEC (P<0.05) and the group without siRNA transfection (P<0.05).3.6×10-8mol/L insulin, with48h significantly inhibit the expression of DDAH1and DDAH2gene and protein in HUVEC (P<0.05), as well as the group without siRNA transfection (P<0.05).4. Transfected with siRNA-SREBP-1and siRNA-SREBP-2 alone can not raise the expression of DDAH1and DDAH2genes and proteins. But the suppression on DDAH1and DDAH2was significantly reduced in the groups with transfection of siRNA-SREBP-1and siRNA-SREBP-2(P<0.05).Conclusions:1. High insulin concentration induces dysfunction on HUVEC, which related with injury;2. High insulin concentration induces protein and gene expression of SREBP-1and SREBP-2, while suppresses DDAH1and DDAH2;3. Silent the expression of SREBP-1and SREBP-2with siRNA reduced the suppression of high insulin concentration on DDAH1and DDAH2expression of gene and protein.4. SREBP/DDAH/ADMA pathway participates the damage effect of high insulin on HUVEC.