Experimental Study On The Mechanism Of Blood Pressure Elevation In The Insulin-Resistant State

On account of endothelial dysfunction's tight association with blood pressure elevation in the insulin-resistant state, people pay more and more attention to it in cardiovascular researches. Insulin signaling plays a key role in the maintenance of vascular endothelial function. In the insulin-resistant state, defects in insulin action lead to endothelial dysfunction. Recent evidence suggested that endothelial dysfunction can be detected early in the spectrum of insulin resistance. So this article focused on the change of endothelial function and the disorder of insulin/phosphatidylinositol 3-kinase (PI3-K) pathway to explore the mechanisms related to blood pressure elevation in the insulin-resistant state, at the level of animal model and cell culture.Objectives (1)To study the mechanisms underlying blood pressure elevation in insulin-resistant rats. (2) To investigate the influences of sucrose-rich diet,L-NAME and renin-angiotensin system on the serum nitric oxide (NO), superoxide anion(O₂^-) in rats and their roles in hypertension pathogenesis. (3)To explore the impact of terahydrobiopterin(BH₄),L-arginine(L-Arg) and local angiotensinII(AngII) formation on endothelial function by observing their effects on human umbilical vein endothelial cells(HUVEC) O₂^- generation. (4) To elucidate the role of insulin/ PI3-K/Akt/endothelial NO synthase signaling pathway in endothelial NO production.Methods In the first part of animal experiments, we established streptozotocin(STZ)-induced diabetic rats and different insulin-resistant animal models by feeding rats with sucrose-rich diet , Nomega-nitro-L-argininne-methylester(L-NAME) or sucrose-rich diet + L-NAME,respectively. Then heart rate, blood pressure and serum endothelial NO synthase(eNOS),NO, superoxide dismutase(SOD), O₂^- as well as plasma AngIIof all rats were measured. In another part, we used two-kidney-one-clip(2KlC) renovascular hypertension rat models and insulin-reistant rats induced by sucrose-rich diet or L-NAME. And at the end of this experiment, all parameters above were tested. At the cell culture level, we investigated the effects of BH4, L-Arg, angiotensin I (AngI) and Irbesartan(Irb) on the production of Ang IK N(X O2 and the influences of insulin, glucose, L-NAME and Wortmannin (inhibitor ofPBK) on the PI3-K expression and NO> O2" generation in HUVEC.Results In the insulin-resistant and STZ-induced diabetic animal models, heart rate is not different from the control group (P>0.05). Systolic blood pressure was higher in sucrose-rich diet,L-NAME, sucrose-rich diet + L-NAME(4 weeks),STZ and sucrose-rich diet + L-NAME(16 weeks) than control(/><0.01,respectively).Furthermore, the Systolic blood pressure of sucrose-rich diet + L-NAME(16 weeks) group was the highest(P<0.01, respectively).The level of NO in sucrose-rich diet(P<0.05),L-NAME(P <0.01), sucrose-rich diet + L-NAME(4 weeks)(/> <0.01) and sucrose-rich diet + L-NAME(16 weeks) (P <0.01) decreased. And the more reduction of NO was seen in sucrose-rich diet + L-NAME(16 weeks) than sucrose-rich diet and sucrose-rich diet + L-NAME(4 weeks) (P <0.01,respectively).When compared with controls, there was an obvious elevation of O2 in sucrose-rich diet, L-NAME, sucrose-rich diet + L-NAME(4 weeks) and sucrose-rich diet + L-NAME(16 weeks) and STZ, whereas a reduction of SOD was observed in sucrose-rich diet, sucrose-rich diet + L-NAME(16 weeks) and STZ. The eNOS activity decreased in L-NAME. We also found an elevation of Ang II in sucrose-rich diet + L-NAME(16 weeks) (P <0.01) and an reduction of Ang II in STZ(P <0.05).In another part of animal experiment, heart rate and systolic blood pressureof renovascular hypertension was higher than that of controls, sucrose-rich diet and L-NAME. When compared with control group, the systolic blood pressure ofL-NAME and sucrose-rich diet elevated significantly (/><0.01). And L-NAME showed higher blood pressure than sucrose-rich diet did. There was no significant difference of Ang II among those groups, respectively (/*>().05).When compared with control group,the level of NO was reduced in L-NAME and sucrose-rich diet. In addition, L-NAME was the lower one^O.Ol). However, an elevation of NO was observed in renovascular hypertension compared with controls, L-NAME and sucrose-rich diet (/><0.01, respectively). The eNOS activity of L-NAME was lowest among the four groups. And there was no significant variation of eNOS in sucrose-rich diet and renovascular hypertension (P>0.05 vs controls). Sucrose-rich diet showed reduced SOD activity compared with control group and renovascular hypertension, L-NAME. The O2" of sucrose-rich diet, L-NAME, renovascular hypertension increased.In the first part of cell culture, 20 u M BH4,15 u M BH4+100 u M L-Arg and 20 u M BH4+IM L-Arg significantly reduced the level of O2 compared with controls(/)<0.01,respectively).However, L-Arg alone have no effect on O2 generation in HUVEC(P>0.05). BH4(10p M ,15 u M ,20 u M),L-Arg(10 u M,100 uM,lmM) and BH4(10uM ,15 uM ,20nM)+L-Arg (10 u M,100u M,lmM) suppressed Ang II production in HUVEC(/*<0.01 vs control, respectively). And the inhibitory effect of BH4(10 u M ,15 u M ,20 u M)+L-Arg (10 u M,100 u M,lmM) was more obvious than BH4 alone or L-Arg alone. BR^lOu M ,15 u M ,20 u M),100 u M L-Arg,lmM L-Arg and BH4(10 u M ,15 u M ,20 u M) +L-Arg (10 u M,100u M,lmM) significantly increased NO in endothelial cells. However, there is no difference between BRtOOn M ,15 u M ,20 u M) +L-Arg (10 u M,100 u M,lmM) and corresponding concentration of BH4 or L-Arg. (/>>0.05). BH4(10 u M ,15 u M ,20 u M) ,L-Arg (10 u M,100 u M,lmM) and BH4(10 u M ,15 u M ,20 n M) +L-Arg (10 u M,100 u M,lmM) elevated the eNOS activity in HUVEC(P<0.01 vs control, respectively). We also observed increased SOD activity induced by BH4(10uM ,15 uM ,20uM),L-Arg (100uM,lmM)and BH4(10uM ,15 uM ,20 u M) +L-Arg (10 u M,100n M,lmM)(/><0.01 vs control, respectively). Cultured HUVEC could convert Ang I to Ang II, so Ang II elevated significantly in the presence of Ang I as compared with control group. And this kind of promoting effects of Ang I was diminished but not deleted by BH4(15n M ,20 u M). The generation of Ang II in the presence of BH4(10n M ,15 u M ,20 u M)+ AngI + Irb was higher than that in control group. AngI ,BH4(10 U M ,15 u M ,20 u M)+ AngI, BH4(10 u M ,15 u M ,20 u M)+ AngI + Irb elevated the activity of eNOS in HUVEC (PO.01 vs control, respectively). And this kind of effect was more obvious in the presence of Angl+ BH420 n M+ Irb than that in the presence of Ang I. When compared with controls, BH4(10 M M ,15 u M ,20 u M)+ AngI, BH4(10 u M ,15 u M ,20 u M)+ AngI + Irb promoted endothelial NO production. SOD activity was elevated by Ang I,AngI+ BH4(15 n M ,20 u M)and Angl+ BH4(15 u M,20 u M)+ Irb, respectively. Ang I increased O2 production in HUVEC as compared with control group. And this effect was corrected by adding BH4(10 u M ,15 u M ,20 u M), BH4(10 u M ,15 u M ,20 u M) + Irb. Furthermore, BH4(10 u M ,15 u M ,20 u M)+ AngI and BH4(10 u M ,15 u M ,20 u M)+ AngI + Irb reduced O2" generation in HUVEC as compared with controls.In the second part of cell culture, insulin (10 mU /L,100 mU /L) significantly increased NO production, eNOS activity and PI3-K expression in HUVEC as compared with control group. And insulin alone had no effect on the generation of O2 and the activity of SOD. In the presence of 25mM glucose+100 mU /L insulin,L-NAME,L-NAME+insulin(10 mU /L,100 mU /L) and Wortmannin, Wortmannin+insulin(10 mU /L,100 mU /L), NO production and eNOS activity significantly decreased(/><0.01 vs control, respectively) accompanied by elevated O2" production(/><0.01 vs control, respectively). When compared with controls, L-NAME+insulin(10 mU /L,100 mU /L) and Wortmannin, Wortmannin+insulin(10 mU /L,100 mU /L) promoted PI3-K expression...