| Objective:In the present study,Goto-Kakisaki（GK）rats and human aortic endothelial cell（HAEC）with insulin resistance（IR）were used to investigate the amelioration and mechanism of nebivolol on vascular endothelial insulin resistance（IR）in type 2diabetic rats,and then to provide new ideas for the prevention of diabetic cardiovascular complications.Methods:（1）Animal experiments:8-week-old,male spontaneous type 2 diabetic GK rats and Wistar rats were selected.Before the experiment,fasting blood glucose（FBG）in GK rats was measured,and GK rats with FBG≥11.1 mmol/L were selected for subsequent experiments.Rats were divided into:Wistar group（equal amount of distilled water,ig）,Wistar+nebivolol group（Wistar+Neb,nebivolol 10 mg/kg/day,ig）,GK group（equal amount of distilled water,ig）,and GK+nebivolol group（GK+Neb,nebivolol10 mg/kg/day,ig）.The experiment lasted for 4 months.During this period,FBG was measured monthly in each group.Fasting insulin（FINS）,oral glucose tolerance test（OGTT）and intraperitoneal insulin tolerance test（IPITT）were measured at the begin and end of the study.Homeostasis model assessment for insulin resistance（HOMA-IR）and insulin sensitivity index（ISI）were calculated.At the end of the study,rats were anesthetized.Then systolic blood pressure（SBP）,diastolic blood pressure（DBP）,heart rate（HR）and mean arterial pressure（MAP）were measured through carotid artery.Blood was collected from the abdominal aorta.And serum nitric oxide（NO）,total cholesterol（TC）,triglycerides（TG）,low-density lipoprotein（LDL）and high-density lipoprotein（HDL）cholesterol were measured.The aorta was rapidly isolated and used for the following experiments:（1）To observe the vascular responses to acetylcholine（ACh）,insulin（INS）,sodium nitroprusside（SNP）and the effect of L-NAME on the relaxation of ACh and INS in vitro.（2）Frozen quickly in liquid nitrogen and stored in-80℃.Western blot was used to measure the expressions of insulin signal pathway-related proteins（IRS-1,p-IRS-1,PI3K,Akt,p-Akt,eNOS,p-eNOS,GLUT4）,endoplasmic reticulum stress-related proteins（ATF6,GRP78,CHOP）and JNK1/2,p-JNK1/2.（2）Cell experiments.1.Establishment of HAEC IR model.In this experiment,two methods were used to choose the appropriate vascular endothelial IR model:30 mmol/L glucose combined with different concentrations of sodium palmitate（SPA,0.1 mmol/L,0.2mmol/L,0.3 mmol/L）for 24 h and 33.3 mmol/L glucose combined with different concentrations of insulin(INS,10-6mol/L,10-7mol/L,10-8mol/L,10-9mol/L)for 48h.Firstly,MTT method was used to test cell activity in order to determine the appropriate method.Secondly,the glucose oxidase method was used to detect the glucose content in culture medium of 33.3 mmol/L glucose combined with different concentrations of INS,then calculating the cellular glucose uptake.Therefore,the appropriate concentration of INS combined with 33.3 mmol/L glucose was determined to induce HAEC IR.2.Effect of different concentrations of nebivolol on the cell activity of HAEC and HAEC IR induced by high glucose and high insulin.Nebivolol（0.1μmol/L,1μmol/L,10μmol/L）was incubated with HAEC and HAEC IR for 48 h,and the cell survival rate of each group was determined by MTT method.3.Effect of different concentrations of nebivolol on glucose uptake and NO concentration of HAEC IR induced by high glucose and high insulin.HAECs were divided into:blank group（Blank,ECM complete medium,no insulin stimulation at the end of the experiment）,control group（Control,ECM complete medium,100nmol/L insulin stimulation at the end of the experiment）,IR group(33.3 mmol/L HG+10-7mol/L INS),different concentrations of nebivolol+IR group（IR group treated with nebivolol 0.1μmol/L,1μmol/L,10μmol/L）.After incubated for 48 h,all groups were starved for 12 h with serum-free culture medium.Except the blank group,cells were stimulated with 100 nmol/L INS for 30 min.TheNO concentration was measured by the total NO kit,and the glucose content was measured by the glucose oxidase method,so as to calculate the glucose uptake.4.Effect of nebivolol on the IRS-1/PI3K/Akt/eNOS pathway in HAEC IR induced by high glucose and high insulin.The HAECs were divided into:blank group,control group,IR group,nebivolol+IR group（IR group treated with nebivolol 1μmol/L）.After incubated for 48 h,all groups were starved for 12 h with serum-free culture medium.Except the blank group,cells were stimulated with 100 nmol/L INS for 30 min.Western blot was used to detect the protein expressions of IRS-1,p-IRS-1,PI3K,Akt,p-Akt,eNOS,and p-eNOS in each group.5.Effect of nebivolol on endoplasmic reticulum stress（ERS）in HAEC IR induced by high glucose and high insulin.HAECs were divided into:blank group,control group,IR group,nebivolol+IR group,IR+TUDCA（IR group treated with TUDCA,500μg/ml,endoplasmic reticulum stress inhibitor）group,nebivolol+IR+TM（IR group treated with nebivolol 1μmol/L and TM 5μg/ml,endoplasmic reticulum stress inducer）group,and TM group（HAEC treated with TM 5μg/ml,endoplasmic reticulum stress inducer）.After incubated for 48 h,all groups were starved for 12 h with serum-free culture medium.Except the blank group,cells were stimulated with 100 nmol/L INS for 30 min.TheNO concentration and glucose content were measured,and then the glucose uptake was calculated.Western blot was performed to detect protein expressions of ERS related proteins（ATF6,GRP78,CHOP）,JNK1/2,p-JNK1/2 proteins and insulin signaling pathway-related proteins（IRS-1,p-IRS-1,PI3K,Akt,p-Akt,eNOS,p-eNOS,GLUT4）.Results:（1）Animal experiment results.1.During the experiment period,FBG of GK rats was higher than that of Wistar rats.At the begin and end of the experiment,there was no significant change in FINS between GK rats and Wistar rats,but HOMA-IR,ISI,AUCOGTTand AUCIPITTwere increased significantly in GK rats,suggesting that GK rats had abnormal glucose tolerance and systemic IR.Nebivolol had no significant effects on the above indexes.At the end of the experiment（4 months）,there was no significant difference in blood pressure in these groups,but serum NO levels decreased in GK rats.Serum TC,TG and LDL levels increased and HDL levels showed a decreasing trend.Nebivolol administration for 4 months significantly improved the above-mentioned dyslipidemia in GK rats.2.The results of isolated vascular perfusion experiments showed that there was no significant difference in the relaxation of ACh(10-9-10-5mol/L)between GK rats and Wistar rats,but the relaxation induced by INS(10-9-10-5mol/L)was significantly attenuated in aorta from GK rats.Relaxation of ACh was significantly inhibited after20 min of preincubation with L-NAME in both Wistar and GK rats.L-NAME reduced the relaxation of INS in the aorta from Wistar rats,but not aorta from GK rats.There was no significant difference in the non-endothelium-dependent relaxation to SNP(10-9-10-5mol/L)in each group.Nebivolol improved the relaxation to INS,but had no effect on the relaxation of ACh in aorta from GK rats.3.Western blot showed that the abnormal protein expressions of IRS-1/PI3K/Akt/eNOS pathway in aorta from GK rats:the protein expressions of IRS-1,PI3K,Akt,p-Akt,eNOS,p-eNOS,GLUT4,p-eNOS/eNOS were decreased,and protein expression of p-IRS-1 and p-IRS-1/IRS-1 were elevated.However,p-Akt/Akt didn’t alter significantly.Moreover,protein expressions of ERS-related proteins（ATF6,GRP78,CHOP）,JNK1,JNK2,p-JNK1,p-JNK2,p-JNK2/JNK2 were increased.Nebivolol ameliorated the abnormal expressions of the above related proteins.（2）Results of cell experiment.1.Choose of HAEC IR model:cell activity concentration-dependently decreased in model of 30 mmol/L glucose combined with different concentrations of SPA,but did not change significantly in model induced by 33.3 mmol/L glucose combined with different concentrations of INS.Therefore,IR induced by high glucose and high insulin were selected.Further,INS(10-6mol/L and 10-7mol/L)significantly reduced glucose uptake in HAEC.In consideration of MTT experiment,33.3 mmol/L glucose combined with INS(10-7mol/L)was finally determined to induce HAEC IR model.2.Different concentrations of nebivolol had no significant effect on the cell survival rate of both HAEC and HAEC IR.3.Nebivolol（1μmol/L and 10μmol/L）increased glucose uptake and NO concentration in HAEC IR.Subsequently,nebivolol（1μmol/L）was selected to explore its mechanism in improving HAEC IR induced by high glucose and high insulin.4.Compared with control group,the protein expressions related to IRS-1/PI3K/Akt/eNOS pathway were abnormal in HAEC IR group:protein expressions of IRS-1,PI3K,Akt,p-Akt,eNOS,p-eNOS were decreased,and protein expression of p-IRS-1 and p-IRS-1/IRS-1 were elevated.But there was no significant difference in the protein expressions of p-Akt/Akt and p-eNOS/eNOS.Nebivolol ameliorated the abnormal protein expressions related to IRS-1/PI3K/Akt/eNOS pathway in HAEC IR group.5.Compared with the control group,glucose uptake and NO concentration were reduced in the IR and TM groups,and the protein expressions related to IRS-1/PI3K/Akt/eNOS pathway were abnormal:protein expressions of IRS-1,PI3K,Akt,p-Akt,eNOS,p-eNOS,GLUT4 and p-Akt/Akt were reduced,and p-IRS-1/IRS-1was elevated.The expressions related to ERS were abnormal:protein expressions of ATF6,GRP78,CHOP increased,and protein expressions of JNK1,JNK2,p-JNK1,p-JNK2,p-JNK1/JNK1,p-JNK2/JNK2 were increased.Nebivolol and TUDCA ameliorated the abnormal expressions of the above related proteins,and the effects of nebivolol were significantly inhibited by TM.Conclusions:1.Nebivolol ameliorated vascular endothelial IR in type 2 diabetic rats by regulating IRS-1/PI3K/Akt/eNOS signal pathway.2.Nebivolol improve vascular endothelial IR in type 2 diabetic rats via inhibiting endoplasmic reticulum stress... |
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