Glucose Oxidation-induced Vascular Contraction And Its Underlying Mechanism

Backgrounds:Vascular complications which are prevalent in glucose metabolism disorders,such as diabetes,are associated with impaired vascular contraction-relaxation function.Long term metabolism disorders(high glucose and high insulin)are proved to damage vascular function through impairing endothelial cells.Meanwhile,dietary intervention,weight loss and physical activity can benefit vascular function.Although emerging evidences show that vascular function is closely related to metabolism,the direct effect of metabolism on vascular contraction-relaxation in physiological or diabetic condition is poorly understood.Glucose metabolism is the major energy source in vascular contraction-relaxation.Although anaerobic glycolysis is regarded as the preferable metabolic model in vascular metabolism,there is a growing body of evidence showing that mitochondria are involved in regulation of vascular function.For example,mitochondrial fission in vascular smooth muscle is proved to induce vascular contraction.However,the role of mitochondria in vascular function regulated by glucose metabolism is unknown.Aims:This study is aimed to explore the effect of glucose metabolism on vascular contraction-relaxation and the role of mitochondria in this phenomenon.Methods and Results:1.We recruited 8 healthy male adults(20-30 years old)and determined blood pressure,cardiac output and the diameter of brachial artery during oral glucose tolerence test.There was a transient hypertension with decreased diameter of brachial artery at 30 min after glucose challenge(1.0 g/kg,oral).However,the cardiac stroke volume,heart rate,cardiac output did not show any change.Relationship analysis showed there was a close relationship between blood glucose and blood pressure(P=0.02),as well as blood glucose and vascular diameter(P=0.03).These results indicated that transient hypertension induced by glucose was attributed to vascular contraction.2.To explore the direct effect of glucose on vascular function,thoracic aorta of mice was isolated.The results showed that vascular contraction was induced by glucose(30 mM)which was not dependent on osmotic pressure.However,30 mM high glucose was not in physiological or most of pathological condition.Based on the fact that insulin secretion was induced by glucose in vivo,we used glucose(10 mM)and insulin(10 nM)mixture(G+I).The results showed that G+I induced the similar extent of vascular contraction induced by glucose(30 mM).It was interesting that the transient contraction induced by G+I was similar with that in vivo but different from the consistent contraction induced by glucose(30 mM)in vitro.3.It is well known that both smooth muscle and endothelium are involved in vascular contraction-relaxation.However,the results showed that there was no change in vascular contraction induced by glucose(30 mM)or G+I with or without endothelium.Then,primary vascular smooth muscle cells(VMSCs)from thoracic aorta of mice were isolated and the phosphorylation level of myosin light chain(MLC),a key protein in vascular smooth muscle contraction,was determined,The results showed that the level of MLC phosphorylation was increased by glucose(30 m M)or G+I.In addition,both glucose(30 mM)-and G+I-induced MLC phosphorylation were inhibited by ?-cyano-4-hydroxycinnamic acid(?-CCA),an inhibitor of the mitochondrial pyruvate transporter,suggesting that glucose oxidation in mitochondria was necessary in glucose-induced vascular contraction.4.Mitochondrion is the glucose metabolism center.In VSMCs,the levels of mitochondrial oxygen consumption and electron donors NADH and FADH2 in G+I group were much more than these in glucose(30 mM)group.Therefore,we used G+I as the glucose oxidation stimulation in the following experiments.To further explore which component of mitochondria affect vascular contraction,we used FCCP(H+ ionophore),Valinomycin(K+ ionophore),Nigericin(H+/K+ ionophore)and Monesin(H+/Na+ ionophore)to find whether mitochondrial membrane potential(??m),proton gradient,ATP,K+ or Na+ was involved in vascular contraction.The results showed there was a same trend between ??m and vascular contraction-relaxation.Then,it was proved by using NH4 Cl and CH3 COONa that ??m increasing induced vascular contraction while ??m decreasing induced vascular relaxation.Furthermore,??m in VSMCs was increased by G+I,which was attenuated by FCCP(decreasing ??m)or Nigericin(maintaining ??m)pretreatment,and the capacity of vascular contraction was also weaken by FCCP or Nigericin pretreatment.These results showed that glucose oxidation induced vascular contraction was mediated by ??m increasing.5.Next,we focused on the specific mechanism how was vascular contractionrelaxation regulated by ??m.There are two signal pathways which regulate MLC phosphorylation,Ca2+ dependent myosin light chain kinases(MLCK)pathway and Ca2+ independent Rho kinase(ROCK)pathway.The contraction capacity was decreased by ROCK inhibitor(Y27632 and Fasudil)rather than Ca2+ signal disruption(BAPTA-AM,TG)which indicated that ROCK played an important part in glucose oxidation induced-contraction.Rho family is the major upstream of ROCK.We found that mitochondrial Rho(miro)rather than RhoA(located in cytoplasm)was activated by glucose oxidation.Silence miro expression by miro-siRNA decreased MLC phosphorylation while overexpression of miro by miro-adenovirus increased MLC phosphorylation following G+I challenge.miro activity was also elevated by ??m hyperpolarization and decreased by ??m depolarization.These results suggested that glucose oxidation-induced vascular contraction was mediated by ??m–miroROCK-MLC pathway.6.To explore whether vascular dysfunction in diabetes was mediated by glucose oxidation-induced vascular contraction,we used STZ and high fat diet-induced rats and db/db mice.The results showed that the levels of blood glucose,insulin and blood pressure were still high while abdominal aorta diameter was decreased at 90 min after glucose challenge(2.0 g/kg,ip.).In isolated thoracic aorta,the contraction duration of db/db mice was much longer than that in WT mice.Meanwhile,??m had a similar change trend with vascular contraction,??m was temporally increased by G+I in WT mice while consistently increased in db/db mice.We also found that phosphorylation levels of MYPT-1 and MLC which are the downstream of ROCK were still high in db/db mice at 30 min which had already returned to normal in WT mice.These results indicated that the increased vascular tone in diabetes might be mediated by sustained ??m increasing.Conclusions: Our main findings: 1.Glucose oxidation induces vascular contraction which is dependent on elevated ??m in VSMCs.2.??m-miro activation induces vascular contraction through ROCK-MLC pathway,and persistence ??m increasing in diabetes mediates sustained vascular contraction.In summary,our study reveals a new metabolic way to regulate vascular contraction,and the disturbance in diabetic condition mediates sustained vascular contraction.These findings provide a new theory to explain the occurrence of diabetic vascular diseases.