Irisin Lowers Blood Pressure By Improvement Of Endothelial Dysfunction Via AMPK-Akt-eNOS-NO Pathway In The Spontaneously Hypertensive Rat

Background and PurposeHypertension is a major public health problem,affecting approximately one billion people worldwide.Exercise,as a nonpharmacological antihypertensive therapy,is able to decrease blood pressure even in subjects with low responsiveness to medical treatment and regular physical exercise is highly recommended by current European and American hypertension guidelines.However,the underlying mechanisms by which exercise decreases blood pressure have not been fully elucidated.Previous studies have provided evidence that endurance aerobic training has an anti-hypertensive effect,which may be caused by a decrease in the activities of the sympathetic and renin-angiotensin systems and enhancement of baroreceptor sensitivity.Additionally,Joham et al have found that aerobic training increases insulin sensitivity.Sun MW et al proposed that moderate levels of exercise enhance vascular endothelial nitric oxide synthase(e NOS)activity resulting in t he improvement of endothelium-dependent vasodilatation.Furthermore,a recent study showed that exercise training could even modulate specific mi RNAs in the heart,artery,and skeletal muscle to reduce blood pressure.The skeletal muscle is an important endocrine organ that can secrete interleukins,tumor necrosis factor ?,leptin,and resistin and many diseases are closely related to its disorder.It has been reported that more than 1000 genes are “activated” by exercise training in human skeletal muscle,all of which may contribute to improvement in health.Recently,a newly found exercise-mediated polypeptide called irisin,the cleavage of extra cellular domain of fibronectin Type III domain-containing 5 protein(FNDC5),has drawn a lot of attention.Exercise can up-regulate transcription factor PPAR? co-activating factor 1?(PGC-1?)which promotes muscle-derived FNDC5 expression and then releases irisin into circulation to increase body energy expenditure.Both FNDC5 and irisin are decreased in patients with type 2 diabetes mellitus and irisin has been reported to be beneficial in glucose homeostasis,insulin resistance,and related morbidities,including obesity.Due to the close relationship between metabolic diseasesand hypertension,it is possible that exercise,via the myogenic factor irisin,may lower blood pressure.Zhang et al and Jiang et al have reported that irisin(0.1-100mM)caused endothelium-dependent and-independent vasodilation of arteries pre-constricted with phenylephrine in mice and rats.Zhang et al also reported that bolus injections(2 min)of high doses of irisin(0.625 to 4mg)decreased the blood pressure of Sprague-Dawley and spontaneously hypertensive rats(SHRs).In humans,the circulating concentration of irisin is 3.6ng/ml in sedentary individuals and increases to 4.3ng/ml in individuals undergoing aerobic interval training.The circulating concentration irisin in rats detected by ELISA is around 300-600ng/ml.Therefore,in order to reflect the physiological effect of irisin,we studied the effect of low doses of irisin on blood pressure and low concentrations of irisin on arterial relaxationin normotensive Wistar-Kyoto(WKY)and SHRs.Mammalian AMP-activated protein kinase(AMPK)is a serine/threonine protein kinase that has been proposed to function as an intracellular energy sensor(sensitive to the AMP/ATP ratio)and is involved in the regulation of cellular and whole body metabolism.NO is one of the most important factors for the relaxation of blood vessels and changes in NO bioavailability affect blood flow and arterial blood pressure.In the vasculature,activation of endothelial AMPK has been shown to phosphorylate e NOS1177,stimulating NO releaseand subsequent vasodilatation of both large conduit and resistance arteries.The endothelium-dependent mesenteric arterial relaxation in mice due to high concentrations(0.1-100mM)of irisin has also been reported to be related to NO-c GMP pathway.Therefore,our present study was designed to determine whether or not the AMPK-eNOS-NO pathway is involved inthe vasorelaxing effect of irisin in SHRs.MethodsPart ?1.WKY rats and SHRs,ranging in age from16-18 weeks,were studied in our experiment.After anesthesia and then tracheotomy were performed,the right carotid artery was catheterized with polyethylene-50 tubing for blood pressure monitoring,one side of external jugular vein was catheterized with polyethylene-50 tubing for irisin injection.After achieving stable hemodynamic conditions and recording of baseline blood pressures for 5min,the rats received an bolus intravenous injection of irisin(0.1,1,10mg/ml)or heat-denatured irisin.2.The vasoreactivityof isolated mesenteric arteries was measured by an isometric Mulvany-Halpern small-vessel myograph(model 91 M620,J.P.Trading,SciencePark,Aarhus,Denmark).Ach(1-100nM)induced endothelium-dependent relaxation was obtained in PHE-pre-constricted segments pre-incubated in absence or presence of irisin and the procedure was repeated with SNP(1-100nM).To study the effect of irisin on vasoconstriction,acumulative concentration-response to PHE(1-10mM)was obtained in arterial segments pre-incubated in absence or presence of irisin.Part ?1.Toconfirm that irisin-mediated increase in NO production,human coronary artery endothelial cells were evaluated of intracellular NO levels with DAF-2DA fluorescence assay and concentrations of NO metabolites nitrite and nitrate in the cell culture supernatant were determined using annitrite and nitrate assay kit.Some assays were performed in the presence of L-NAME(100mM)throughout the experimental period.2.We evaluated the effect of different centration(100,200,300,600ng/ml)of irisin which was treated for different time(15min?30min?1hr?3hr?6hr)on eNOS-ser1177 phosphorylation(p-e NOS)levels in rat mesenteric arteries from SHRs and human coronary endothelial cells through immoblotting.Besides,we also assessed the effects of irisin on n NOS phosphorylation in human aortic endothelial cells.3.To further confirm the effect of irisin on NO production is physiologically significant,NOS inhibitor L-NAME(100mM),COX inhibitor indomethacin(10mmol/L)and EDHF inhibitor KCl(60m M)were studied to evaluated their effcet on the synergistic vasorelaxant effect of irisin and Ach.Moreover,NOS inhibitor L-NAME were used to observe its effect on the blood pressure-lowering effect of irisin in SHRs.Part ?In-vitro study,to elucidate the mechanisms underlying the increase in e NOS phosphorylation in response to irisin,AMPK and Akt,the upstream transducers of e NOS phosphorylation,were evaluated by immoblotting.In addition,AMPK inhibitor Compound C(20mM)was pretreated for 30 min to observe its effect on Akt and e NOS phosphorylation level and on the synergistic vasorelaxant effect of irisin and Ach.ResultsPart ?:Irisin lowered blood pressure by improvement of endothelial dysfunction in SHRs.1.Irisindecreased blood pressure in a dose-dependent(0.1,1,10mg/kg)mannerin SHRs.By contrast,in WKY rats,irisin had no effect on blood pressure.In SHRs,the bolus intravenous injection of irisin(10mg/kg)started to decrease blood pressure after 5min,reached significance after 10 min,with the maximum effect noted after 20min;the vasodepressor effect of irisin was no longer evident at 90 min.Heat-denatured irisin had no effect on blood pressure.Irisin,also,had no effect on heart rate.2.Irisin(3000ng/ml),by itself,had no direct vasorelaxant effect in mesenteric arteries from SHRs and WKY rats,pre-constricted with PHE.However,it augmented the Ach-mediated vasorelaxation in mesenteric arteries from SHRs but not WKY rats.We also found that irisin could decrease the vasoconstriction induced by PHE in the mesenteric artery of SHRs.We found that there was no additive effect of irisin on SNP-induced vasorelaxation in mesenteric arteries from both SHRs and WKY rats.Those results indicate that the vascular dysfunction in SHRs can be ameliorated by irisin in an endothelium-dependent mechanism.Part ? : Irisin-mediated increase in NO production decreased endothelial dysfunction in the mesenteric artery of the SHR.1.Irisin increased NO production,measured by DAF-2 DA fluorescencestaining,in a time-and concentration-dependent manner.L-NAME(100mM),a NOS inhibitor,completely abrogated the irisin-induced increase in NO production.When endothelial cells were treated with irisin(3000ng/ml [240nM])for 10 min,nitrite and nitrate increased,which was also significantly inhibited by pretreatment with L-NAME(100mM)implying that the effect was dependent on the activation of NOS.In additional studies,human coronary endothelial cells were pre-incubated with irisin(3000ng/ml)for 1hr,washed with HEPES buffer,and then treated with Ach(100nM).We found that irisin(3000ng/ml)increased the ability of Ach(100n M)to increase NO production after 240 s of incubation.2.In the mesenteric arteries,as compared with controls,irisin(600ng/ml)incubation for 30 min significantly stimulated e NOS-ser1177 phosphorylation_as early as 15 min,peaked at 60 min and then gradually decreased close to the basal level at 240 min.Irisin(30min incubation)also increased eNOS-ser1177 phosphorylation_in a concentration-dependent manner.Irisin(600ng/ml)incubation also stimulated eNOS-ser1177 phosphorylation in human coronary endothelial cells similar to the rat mesenteric arteries but the effect occurred later,i.e.,30 min.Although the expression of nNOS was weaker than eNOS,irisin,at 600ng/ml concentration,also increased nNOS phosphorylation at 60 min.3.The effect of irisin on NO production is physiologically significant,because the synergistic vasorelaxant effect of irisin and Ach was blocked by the NOS inhibitor,L-NAME(100mM)but not by inhibitors of COX and EDHF,indomethacin(10mmol/L)and KCl(60mM),respectively.Moreover,the blood pressure-lowering effect of irisin in SHRs was almost completely blocked by pretreatment with L-NAME(30mg/kg,bolus injection).Part ?:Irisin phosphorylates eNOSthrough up-regulation of AMPK and Akt phosphorylation in human coronary endothelial cellsIrisin increased AMPK(Thr172)and Akt(Ser473)phosphorylation in a concentrationand time-dependent manner,but had no effect on total AMPK and Akt.An additiona l study showed that in the presence of Compound C(20mM),an AMPK inhibitor,the irisin-mediated increase in the phosphorylations of Akt and e NOS were blocked.Moreover,pre-treatment with Compound C partially blocked the synergistic vasorelaxant effect of irisin and Ach.ConclusionsIn summary,we found that low doses or physiological concentrations of irisin did not lower blood pressure or dilate the mesenteric artery of WKY rats.By contrast,irisin decreased blood pressure of SHRs in a concentration dependent manner.Physiological concentrations of irisin(48 n M and 240 n M)did not dilate the mesenteric artery of SHRs pre-contracted by PHE.However,the same concentration of irisin ameliorated the impaired-endothelial relaxation response to Ach in the mesenteric artery of the SHR.The vasodilatory effect of irisin might be caused by the stimulation of arterial endothelial cells to increase AMP/ATP levels and NO release by activation of AMPK and Akt.The up-regulation of NO production improved the endothelial dysfunction in the SHR and ultimately decreased blood pressure,which may be helpful to normalize the high blood pressure of hypertensive patients.