Protective Effects Of Glucagon Like Peptide-1Via A CAMP/PKA/Rho Dependent Signal Pathway On Cardiac Microvessels Injury In Diabetes

BackgroundDiabetes mellitus （DM） is recognized as a major risk factor for cardiovascular disease,the leading etiology of morbidity and mortality in the diabetic population. Diabeticcardiovascular disease results from many causes such as microangiopathy, myocardialmetabolic abnormalities and fibrosis. Under microangiopathy, the vessel wall ofmicrovessels become thicker and vulnerable for bleeding, protein leakage, and slow bloodflow. Accumulating evidence has demonstrated that microvascular injury plays a veryimportant role in the diabetic cardiovascular dysfunction. However, there are still feweffective strategies to prevent the progress of the microvascular dysfunction in diabetesmellitus. Glucagon-like peptide-1（GLP-1） is a hormone predominately synthesized andsecreted by intestinal L-cells. Pharmacological modulation of the GLP-1system hasemerged as a treatment option for diabetes mellitus. In addition to its glucose loweringproperties, GLP-1was found to have multiple cardioprotective effects. Impaired cardiacmicrovascular function is thought to contribute greatly to the diabetes cardiovasculardisease. Yet the effects of GLP-1on cardiac microvessels remained unclear, this studywas to assess if GLP-1could protect the cardiac microvessels and subsequently improvecardiac function and glucose metabolism and to investigate the underlying regulatorymechanism in diabetes.Methods1. Male Sprague-Dawley （SD） rats （weight,200-220g） were made diabetic usingstreptozotocin （STZ） injection. Blood glucose levels were tested1week after STZinjection. Animals with glucose levels≥16.7mmol/L were considered diabetic rats.2. To determine the dosage of insulin to control blood glucose in DM group at the similarlevel as vildagliptin-and exenatide-treated groups, diabetic rats were randomized intothe following groups:（1） Vildagliptin group that received daily treatment ofvildagliptin at1mg/kg of body weight;（2） Exenatide group that received dailytreatment of exenatide at1nmol/kg of body weight;（3） Insulin group that receiveddaily treatment of insulin at0.5U,1U,1.5U, and2U. Blood glucose was measuredonce a day for2weeks.3. STZ-induced diabetic rats were randomized to12weeks of treatment with vehicle,vildagliptin （DPP-4inhibitor,1mg/kg/d）, exenatide （GLP-1analogue,1nmol/kg/d）,or insulin （1.5U）. Before and after treatment, blood glucose levels, weight, and plasmainsulin levels were assessed.4. Cardiac microvascular barrier function was detected by scanning electron microscopyand transmission electron microscopy.5. Cardiac function was examined by echocardiographic measurements.6. Cardiac glucose metabolism was examined by18F-FDG PET/CT. 7. Adult rat cardiac microvascular endothelial cells （CMECs） were isolated and replacedto different conditions after confluence: normal glucose medium （5.5mmol/L）, normalglucose plus GLP-1（10nmol/L）, high glucose medium （25mmol/L） plus vehicle（DMSO）, high glucose plus GLP-1（10nmol/L）.8. Glucagon-like peptide-1receptor （GLP-1R） expression was detected byimmunofluorescence stainning and Western blot.9. Superoxide assay kit and dihydroethidine （DHE） staining were used to assessoxidative stress.10. TUNEL staining and caspase3expression were used to assess the apoptosis ofCMECs.11. H89was used to inhibit cAMP/PKA pathway; fasudil was used to inhibit Rho/ROCKpathway. The protein expression of Rho, ROCK, p47^phox, gp91^phox, p22^phox, and p40^phoxwere examined by Western blot analysis.Results1. Insulin treatment at1.5U dosage per day had the similar effect in blood glucose tothose elicited by vildagliptin and exenatide in diabetic rats.2. Significant deficit in barrier function of cardiac microvessels was found in diabeticrats. After12weeks of treatment with vildagliptin or exetinade, the cardiacmicrovascular barrier function was improved, in a manner more pronounced than theinsulin.3. Diabetes led to a defective18F-FDG uptake in the heart, the effect of which wassignificantly improved by vildagliptin or exenatide treatment. Insulin treatmentexhibited less18F-FDG uptake in the heart compared with administration ofvildagliptin or exenatide.4. Diabetic rats exhibited significantly dampened diastolic function, as manifested byincreased E/A ratio and LVEDD, the effects of which were mitigated by vildagliptin orexenatide treatment. Compared with insulin treated group, vildagliptin or exenatide further improved cardiac diastolic function in diabetic rats. However, there was littledifference in FS among the four groups studied.5. GLP-1R was expressed on CMECs.6. High glucose increased ROS production and NADPH activity after24hoursincubation in CMECs, while GLP-1decreased high glucose-induced ROS production.The protein expression of p47^phox, gp91^phox, p22^phox, and p40^phoxsubunits of NADPHoxidase were significantly increased in high glucose-induced CMECs compared withcontrol group. After treated with GLP-1, the expression of p47^phox, gp91^phox, p22^phox,and p40^phoxwere significantly decreased compared with high glucose-inducedCMECs.7. GLP-1exerted an anti-apoptotic effect on high glucose-induced CMECs.8. Incubation of CMECs with high glucose significantly upregulated Rho expression andelicited a significant increase in ROCK expression. Treatment of CMECs with GLP-1significantly alleviated high glucose-induced increase in Rho and ROCK expressionwithout affecting these parameters under normal glucose condition. Incubation of cellswith the PKA selective inhibitor H89abrogated GLP-1-induced effect on Rhosuppression.9. Chronic exposure of CMECs to high glucose promoted intracellular ROS levels, theeffect of which was obliterated by GLP-1. Incubation of CMECs with fasudil inhibitedROS production. However, combination of GLP-1and fasudil failed to furtherattenuate ROS production.10. The inhibitory effect of GLP-1on high glucose-induced ROS accumulation wassignificantly attenuated by treatment with H89.11. In line with the changes in intracellular ROS accumulation, the inhibitory effect ofGLP-1on high glucose-induced activation of p47^phoxand gp91^phoxwas reduced byH89. The combined treatment with GLP-1and fasudil failed to produce any additiveeffects on high glucose-induced p47^phoxand gp91^phoxexpression and oxidative stress. ConclusionsGLP-1could protect the cardiac microvessels against oxidative stress, apoptosis andthe resultant microvascular barrier dysfunction in diabetes rats, en route to improvedcardiac diastolic function and cardiac glucose metabolism. The protective effects ofGLP-1are dependent on downstream inhibition of Rho through a cAMP/PKA-dependentmanner, resulting in a subsequent decrease in the expression of NADPH oxidase. Thesefindings should provide important implications for diabetes patients with cardiovasculardisease, where GLP-1may hold promise for prevention and treatment.