Effect Of Endoplasmic Reticulum Stress On Diabetes Induced Vascular Dysfunction

Background: Cardiovascular complications are the most common causes leading to disability and death among diabetic patients. Diabetes mellitus is considered to be a risk factor of hypertension, but the molecular mechanism is unclear. It was showed that ER stress widely existed in a variety of tissues and organs of diabetes, and was closely related to metabolic disease. It was showed that ER stress played an important role in cardiovascular diseased, including diabetic cardiovascular complications, atherosclerosis and heart failure. The aim of this study is to examine whether ER stress is involved in hyperglycemia-induced hypertension and the possible molecular mechanism. The microvascular vessels from diabetic/prediabetic rats were collected since vascular injury is the pathological basis of cardiovascular disease.Methods: Animal models:(1).Male SD rats were injected with 35mg/kg of STZ intraperitoneally. The rats with non-fasting glucose levels higher than 16.7 mM and lower than 26.0mM were selected as hyperglycemia(STZ group) for further study, while rats in control group were injected with citrate buffer. Body weight and blood glucose levels were monitored. Blood pressure(BP) was measured 1 months later. Transcriptional levels of ER stress marker and UPR related proteins(GRP78, Perk, eif2α, ATF6 and XBP1) in mesenteric resistant arteries(MRA) were measured by RT-PCR; Protein levels of p-eNOS, eNOS, p-IRS1, IRS1, p-Akt, Akt were evaluated by Western blot.(2).Diabetic rats from the part I mated with normal female SD rats, while normal male rats mated with normal female rats. Then the male offspring were chosen for the experimental group(DM-O) and Control group(Con-O) respectively. After birth, body weight, random blood glucose and fasting blood glucose were monitored. Glucose tolerance test(GTT) and blood pressure measuring were performed. The rats in DM-O group were divided into two groups: Treated with ER stress inhibitor — 4-phenyl butyric acid(4-PBA) for 1 month as DM-O-PBA group and treated with PBS as DM-O-PBS group. Animals were sacrificed, MRA and liver samples were then collected. Protein levels of p-Perk, Bip, p-eif2α, eif2α, p-eNOS, eNOS, p-Akt, Akt, p-IRS1, IRS1 were evaluated by Western blot. The ultra microstructure of cells in liver tissue was observed under transmission electron microscope.Results: 1).STZ treatment significantly increased blood glucose levels compared with control group(Con)(P<0.01). Body weight of STZ treated rats was significantly lower than that of Con group(P<0.05). The systolic BP(P<0.05), diastolic BP(P<0.01) and mean BP(P<0.01) in STZ group were significantly higher than those in Con group. The mRNA levels of GRP78, Perk, eif2α, ATF6 and XBP1 in the MRA of STZ group were significantly up-regulated, while STZ injection did not alter the mRNA level of CHOP. Western blot results showed that GRP78 protein levels in the STZ group were significantly higher than that in Con group(P<0.05), and the phosphorylated components of insulin signaling pathways in the STZ group were significantly decreased(p-IRS1/IRS1: P<0.05; p-Akt/Akt: P<0.05); Finally, p-eNOS/eNOS was also reduced(P<0.05). 2).Body weights of offspring of paternal diabetic rats(DM-O) were significantly higher than that of the offspring of control rats(CT-O). There were no significant increase both fasting and random blood glucose levels in DM-O group compared with Con-O group. However, the GTT result showed that glucose tolerance was impaired in the DM-O rats. There was no significant difference of blood pressure between the tow groups. The ultra microstructure of liver cells in DM-O group showed endoplasmic reticulum swelling suggesting ER stress. Western blot showed that the ER stress markers in the MRA of DM-O were significantly higher than that of Con-O. The key protein levels of insulin signaling pathways of p-IRS1/IRS1,p-Akt/Akt ratio in the MRA of DM-O was significantly lower than that in CT-O. Finally, p-eNOS/eNOS ratio was also reduced. However, after receiving 4-PBA, ER stress biomarker expression levels were decreased, and the activities of IRS1, Akt and eNOS were increased.Conclusion: Disturbance of blood glucose induced microvascular dysfunction through IRS1/Akt/eNOS pathway in SD rats, possibly mediated by ER stress.