Role Of GLP-1 In The Regulation Of MLC Phospholylation Induced By Advanced Glycation End Products In Vascular Endothelial Cell

BackgroundThe rapidly increasing incidence of diabetes mellitus (DM) worldwide has been a worldwide health and economic problem. The detrimental effects of diabetes are the high incidence of morbidity and mortality resulted from diabetic complications, especially macro-and micro vascular diabetic complications. The accelerated formation and accumulation of advanced glycation end products (AGEs) in vessels play an important role in the development of diabetic angiopathy. AGEs are a heterogeneous group of modified molecular species formed by nonenzymatic glycation of free amino groups of proteins, lipids, or nucleic acids, which alter cellular function. Interaction of AGEs with its receptor (RAGE) also activates multiple signal transduction pathways and aggravates the development of vascular disease.Vascular endothelial cell (EC) serves as semi-permeability barrier between blood and tissue fluid. The exchange materials in exterior and interior of blood must be passing through vascular endothelial cell. The maintenance of ECs integrality and function is pretty crucial for the normal physiological function. The damage of ECs caused by AGEs formation, oxidative stress and inflammation is an early hallmark of diabetic vasculopathy. Subsequently, endothelial barrier injury occurs with increased cell contractility and destruction of cell junction structure. Cell contractility is mainly associated with myosin light chain phosphorylation mediated cytoskeleton reorganization.Glucagon-like peptide-1 (GLP-1), an incretin hormone, secreted postprandially by the intestinal L-cells, not only modulates the glucose homeostasis in multiple tissues and cells, but also exerts potentially biological effects in cardiovascular systems. It has been reported that GLP-1 exerts protective effects on endothelium-dependent dilatation by suppressing oxidative stress and inflammation. However, whether GLP-1 exerts protective effects on endothelial barrier function and the underlying mechanisms are still not well defined.ObjectiveIn this project, we aim to observe the effect of GLP-1 on the aortic permeability in diabetic rats and investigate whether RAGE/Rho/ROCK signal pathway and MLC phosphorylation in aorta are the target of GLP-1. Furthermore, we also aim to investigate the effect of GLP-1 on the cytoskeleton reorganization of AGEs incubated ECs and elucidate the underlying mechanisms associated with MLC phosphorylation.Methods1. We established diabetic rats model by using streptozotocin injection and high-fat diet. The diabetic rats were given sitagliptin (10mg/kg/d and 30mg/kg/d) for 12 weeks. We made serum biochemical analysis by elisa method, radioimmunoasssay method and enzymatic method.We observed morphology of aorta by using electron microscope and hematoxylin and eosin method. The thoracic aorta rings were made to measure isometric contractile tension. Surface biotinylation technique was used to evaluate permeability. Several proteins expression and phosphorylation were measured by immunohistochemistry and western blot.2. HUVECs were cultured in DMEM at 37? in a humidified atmosphere of 5% CO2. GLP-1, exendin(9-39), anti-RAGE IgG, forskolin, SQ22536, H89, LPA and fasudil were added in culture media respectively or pairedly. The distribution of F-actin was observed by laser scanning confocal microscope. Rho activity was detected by G-LA reagent kit. Several proteins expression and phosphorylation were measured by immunohistochemistry and western blot to investigate whether GLP-1 inhibits Rho/ROCK induced MLC phosphorylation and cell cytoskeleton reorganization via GLP-1R mediated cAMP/PKA signal pathways.Results1. The type 2 diabetic rat model was successfully established. Serum fasting glucose, lipid, insulin resistance and ET-1 were significantly increased and weight, plasma GLP-1 and serum NO levels were significantly decreased in diabetic rats. Serum fasting glucose, insulin resistance and ET-1 levels in diabetic rats were significantly decreased and weight, plasma GLP-1 and serum NO levels in diabetic rats were significantly increased after sitagliptin treatment for 12 weeks.2. Diabetes significantly increased aortic endothelial dysfunction and endothelial permeability. Aortic endothelial dysfunction and endothelial permeability in diabetic rats were significantly attenuated after administration of high dose of sitagliptin for 12 weeks.3. The expression of GLP-1 R in diabetic aorta was significantly decreased. However, the expressions of RAGE, ROCK and MLCK were significantly increased in diabetic aorta. MYPT as well as MLC phosphorylation and Rho activity were also significantly increased in diabetic aorta. Sitagliptin treatment significantly increased GLP-1 R expression and decreased RAGE, ROCK, MLCK expressions in diabetic aorta. MYPT as well as MLC phosphorylation and Rho activity were also significantly decreased in diabetic aorta after administration of sitagliptin for 12 weeks.4. Continuous F-actin was mainly distributed in the cortical area of ECs under normal conditions, forming a typical peripheral actin rim. Few stress fibers were observed. Exposure of ECs to AGE-BSA (200mg/1) induced a shift in F-actin distribution from a web-like structure to polymerized stress fibers. Anti-RAGE IgQ forskolin, fasudil and GLP-1 could reverse the shift of F-actin distribution caused by AGE-BSA. Exendin (9-39), SQ22536, H89 and LPA could significantly block the effect of GLP-1 on F-actin distribution.5. Exposure of HUVECs to AGE-BSA induced a significant decrease of GLP-1R expression and elevation of RAGE, ROCK, MLCK expressions and MYPT, MLC phosphorylation as well as RhoA activity in a dose-and time-dependent manner.6. Anti-RAGE IgG significantly increased GLP-1 expression and decreased RAGE, ROCK, MLCK expressions in HUVECs incubated with AGE-BSA. MYPT and MLC phosphorylation and Rho activity were also significantly decreased in HUVECs incubated with AGE-BSA and Anti-RAGE IgG7. The expressions of RAGE, ROCK and MLC phosphorylation as well as RhoA activity were down-regulated in HUVEC incubated with AGE-BSA when pretreated with forskolin.8. The MYPT and MLC phosphorylation as well as RhoA activity were down-regulated in HUVEC incubated with AGE-BSA when pretreated with fasudil.9. GLP-1 decreased the expressions of RAGE, ROCK, p-MYPT, p-MLC and RhoA activity, and increased GLP-1 R and p-PKA expressions in HUVEC incubated with AGE-BSA. Exendin (9-39), SQ22536, H89 and LPA could significantly block the effect of GLP-1 on these proteins expression.Conclusions1. The permeability of aorta intima in diabetic rats was significantly increased. F-actin of endothelial cells incubated with AGE-BSA was rearrangement. We speculate that these phenomenon were due to activation of Rho/ROCK induced MYPT and MLC phosphorylation caused by the effect of AGE/RAGE mediated signal pathways.2. Attenuation of aorta intima permeability in diabetic rats and endothelial mono layer permeability in AGE-BSA incubated ECs by increasing GLP-1 level may be due to the inhibition of RAGE/ROCK/MLC signaling pathway mediated by GLP-1 administration.3. The possible mechanisms of GLP-1 in the regulation of ECs contractility and vascular permeability in diabetes may be due to the participation of GLP-1R/cAMP/PKA activation and subsequently RAGE/Rho/ROCK inactivation in the barrier stabilizing effect of GLP-1 imposed on AGE-induced ECs.