Wang, Gang Xu, Through a Glucagon -Like Peptide 1-Dependent Mechanism Dipeptidyl Peptidase 4...

Yao and Yu HuangYunfei Pu, Zhiming Zhu, Aimin Xu, Karen S.L. Lam, Zhen Yu Chen, Chi Fai Ng, Xiaoqiang

Limei Liu, Jian Liu, Wing Tak Wong, Xiao Yu Tian, Chi Wai Lau, Yi-Xiang Wang, Gang Xu,Dependent MechanismLike Peptide 1Through a Glucagon

Dipeptidyl Peptidase 4 Inhibitor Sitagliptin Protects Endothelial Function in Hypertension

Print ISSN: 0194-911X. Online ISSN: 1524-4563 Copyright 2012 American Heart Association, Inc. All rights reserved.

is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Hypertension doi: 10.1161/HYPERTENSIONAHA.112.195115

2012;60:833-841; originally published online August 6, 2012;Hypertension.

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Dipeptidyl Peptidase 4 Inhibitor Sitagliptin ProtectsEndothelial Function in Hypertension Through aGlucagonLike Peptide 1Dependent Mechanism

Limei Liu, Jian Liu, Wing Tak Wong, Xiao Yu Tian, Chi Wai Lau, Yi-Xiang Wang, Gang Xu,Yunfei Pu, Zhiming Zhu, Aimin Xu, Karen S.L. Lam, Zhen Yu Chen, Chi Fai Ng,

Xiaoqiang Yao, Yu Huang

AbstractSitagliptin, a selective dipeptidyl peptidase 4 inhibitor, inhibits the inactivation and degradation of glucagon likepeptide 1 (GLP-1), which is used for the treatment of type 2 diabetes mellitus. However, little is known about the roleof GLP-1 in hypertension. This study investigated whether the activation of GLP-1 signaling protects endothelialfunction in hypertension. Two-week sitagliptin treatment (10 mg/kg per day, oral gavage) improved endothelium-dependent relaxation in renal arteries, restored renal blood flow, and reduced systolic blood pressure in spontaneouslyhypertensive rats. In vivo sitagliptin treatment elevated GLP-1 and GLP-1 receptor expressions, increased cAMP level,and subsequently activated protein kinase A, liver kinase B1, AMP-activated protein kinase- and endothelial NOsynthase in spontaneously hypertensive rat renal arteries. Inhibition of GLP-1 receptor, adenylyl cyclase, protein kinaseA, AMP-activated protein kinase-, or NO synthase reversed the protective effects of sitagliptin. We also demonstratethat GLP-1 receptor agonist exendin 4 in vitro treatment had similar vasoprotective effects in spontaneouslyhypertensive rat renal arteries and increased NO production in spontaneously hypertensive rat aortic endothelial cells.Studies using transient expressions of wild-type and dominant-negative AMP-activated protein kinase-2 support thecritical role of AMP-activated protein kinase- in mediating the effect of GLP-1 in endothelial cells. Ex vivo exendin4 treatment also improved endothelial function of renal arteries from hypertensive patients. Our results elucidate thatupregulation of GLP-1 and related agents improve endothelial function in hypertension by restoring NO bioavailability,suggesting that GLP-1 signaling could be a therapeutic target in hypertension-related vascular events. (Hypertension.2012;60:833-841.) Online Data Supplement

Key Words: dipeptidyl peptidase 4 endothelium-dependent relaxation glucagon-like peptide 1 NO protein kinases spontaneously hypertensive rats

Hypertension is caused by pathological changes in renaland vascular structure and function involved in bloodpressure regulation.1 Hypertension can cause renal damage ifit is not properly controlled.2 The impaired vasodilatorresponse is a risk factor for renal function loss in patients withessential hypertension.3 Persistent hypertension alters func-tional characteristics of vascular endothelial cells and isassociated with impaired vasodilatory function.4 Diminishedproduction and function of endothelium-derived NO leads toendothelial dysfunction,5 a crucial initial step culminating invascular events in hypertension.

Dipeptidyl peptidase 4 (DPP-4), also known as CD26, is aubiquitous enzyme detectable in the endothelium.6 Glucagon-

like peptide 1 (GLP-1) produced by L-type cells in theintestine, is a substrate for DPP-4.7 GLP-1 improves glucoseuse in patients with type 2 diabetes mellitus by increasinginsulin secretion and inhibiting glucagon secretion.8,9 Sita-gliptin, a highly selective DPP-4 inhibitor,10 inhibits theinactivation and degradation of GLP-1,11 which is used forthe treatment of type 2 diabetes mellitus as monotherapyor in combination with other antiglycemic agents, such asmetformin.12

The effect of GLP-1 on blood pressure has been reported inboth animal and human hypertension.13,14 Treatment withGLP-1 receptor (GLP-1R) agonists leads to a transient bloodpressure increase attributed to the impact on sympathetic

Received March 13, 2012; first decision March 20, 2012; revision accepted July 11, 2012.From the Institute of Vascular Medicine and Li Ka Shing Institute of Health Sciences (L.L., J.L., W.T.W., X.Y.T., C.W.L., X.Y., Y.H.), Departments

of Imaging and Interventional Radiology (Y.-X.W.), Medicine and Therapeutics (G.X.), and Surgery (C.F.N.), and School of Life Sciences (Z.Y.C.),Chinese University of Hong Kong, Hong Kong, China; Department of Physiology and Pathophysiology (L.L.), Peking University Health Science Center,Beijing, China; Department of Hypertension and Endocrinology (Y.P., Z.Z.), Daping Hospital, Third Military Medical University, Chongqing, China;Departments of Medicine (A.X., K.S.L.L.) and Pharmacology and Pharmacy (A.X.), University of Hong Kong, Hong Kong, China.

The online-only Data Supplement is available with this article at http://hyper.ahajournals.org/lookup/suppl/doi:10.1161/HYPERTENSIONAHA.112.195115/-/DC1.

Correspondence to Yu Huang, School of Biomedical Sciences, Chinese University of Hong Kong, Hong Kong, China. E-mail [email protected],and Limei Liu, Department of Physiology and Pathophysiology, Peking University Health Science Center, Beijing, China. E-mail [email protected]

2012 American Heart Association, Inc.Hypertension is available at http://hyper.ahajournals.org DOI: 10.1161/HYPERTENSIONAHA.112.195115

833 at Chinese University of Hong Kong on August 30, 2012http://hyper.ahajournals.org/Downloaded from

outflow.15 However, continuous infusion of GLP-1 producesa small but insignificant reduction of blood pressure inpatients with type 2 diabetes mellitus.16 Moreover, chronicadministration of recombinant GLP-1 prevents the develop-ment of hypertension and improves endothelial function inDahl salt-sensitive rats.17 By contrast, the effect of DPP-4inhibition on blood pressure is largely unknown, althoughlimited studies show that DPP-4 inhibition by sitagliptinproduces a small blood pressurelowering effect in nondia-

betic patients with hypertension on stable antihypertensivetherapy.18 The present study investigated whether DPP-4inhibition could ameliorate endothelial dysfunction in renalarteries from hypertensive animals and patients, as well aspossible signaling mechanisms involved.

Materials and MethodsA supplemental Methods section can be found in the online-onlyData Supplement.

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Figure 1. Sitagliptin lowers blood pressure and increases renal blood flow in spontaneously hypertensive rats (SHRs). A, Systolic bloodpressure (SBP) in vehicle and sitagliptin-treated Wistar-Kyoto rats (WKYs) and SHRs. B, Renal blood flow in WKY vehicle (1) rats or invehicle (3) and sitagliptin-treated SHRs (2). Data are meanSEM. *P0.05 vs WKY vehicle, #P0.05 vs SHR vehicle. n4.

834 Hypertension September 2012


Measurement and Analysis of Renal Blood Flowby MRI Acquisition ProcedureMRI studies were performed using a 3T clinical whole-body imagingsystem (Achieva, Philips Healthcare, Best, the Netherlands).19

Renal Artery Preparation and Functional StudiesRats were euthanized by CO2 suffocation, and renal interlobararteries were removed and placed in ice-cold Krebs solution. Arterieswere prepared, and changes of isometric tension were recorded.20,21

Western Blot AnalysisProtein expression levels of GLP-1, GLP-1R, phospho-PKA C(protein kinase A catalytic subunit, Thr197), phospho-LKB1 (liverkinase B1, Ser334), phospho-endothelial NO synthase (eNOS;Ser1177), phosphoAMP-activated protein kinase (AMPK; Thr172),PKA C, LKB1, eNOS, AMPK, and AMPK2 were detected byWestern blotting.

NO MeasurementIntracellular NO production was monitored using a fluorescent NO indica-tor 4-amino-5-methylamino-2=,7=-difluorofluorescein diacetate22 and theTotal Nitric Oxide Assay kit.

Data AnalysisResults represent meanSEM from different rats or patients. Statis-tical significance was determined by 2-tailed Student t test or 1-wayANOVA followed by the Bonferroni post hoc test when 2treatments were compared. P values 0.05 indicate statisticallysignificant difference.

ResultsSitagliptin Treatment Lowers Blood Pressure andIncreases Renal Blood Flow in SpontaneouslyHypertensive RatsAmbulatory arterial pressure in conscious, unrestrained spon-taneously hypertensive rats (SHRs) was monitored by radio-telemetry. The ambulatory systolic blood pressures (SBP)were significantly lower in 2-week sitagliptin-treated SHRscompared with vehicle-treated SHRs (averaged SBP, 1608versus 1805 mm Hg; n4 each group), whereas sitaglitintreatment did not alter SBPs in Wistar-Kyoto rats (WKYs;averaged SBP, 1204 versus 1194 mm Hg; n4 eachgroup; Figure 1A). This is further confirmed by the direct

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Figure 2. Endothelial function improvedby sitagliptin is via restoring endothelialNO synthase (eNOS) activity in sponta-neously hypertensive rat (SHR) renalarteries. A, Endothelium-dependentrelaxation (EDR) in renal arteries fromvehicle and sitagliptin-treated Wistar-Kyoto rats (WKYs) and SHRs. B,Endothelium-independent relaxations tosodium nitroprusside (SNP) in renalarteries from vehicle and sitagliptin-treated rats. Phosphorylations of proteinkinase A catalytic subunit (PKA C) atThr197 (C), LKB1 (liver kinase B1) atSer334 (D), AMP-activated protein kinase(AMPK) at Thr172 (E), and eNOS atSer1177 (F) in renal arteries from vehicleand sitagliptin-treated WKYs and SHRs.Data are meanSEM. *P0.05 vs WKYvehicle, #P0.05 vs SHR vehicle. n10for acetylcholine (ACh)-induced relax-ations; n4 for SNP induced relaxations;n6 for Western blotting.

Liu et al DPP-4 Inhibition Restores Endothelial Function 835


measurement of SBP with a direct catheter in anesthetizedrats (Figure S1A, available in the online-only Data Supple-ment) and the tail-cuff method (Figure S1B). However,sitagliptin treatment did not significantly lower mean arterialblood pressure or diastolic blood pressure in all groups ofrats. It is noted that the effect of sitagliptin on SBP inSHRs was a step decrease that occurred between days 3and 4 and that there was no diurnal rhythm in control andsitagliptin-treated rats (Figure S1C). Renal blood flow(RBF) reduction in SHRs was restored by 2-week sitaglip-tin therapy (Figure 1B).

Sitagliptin Improves Endothelial Function in SHRRenal ArteriesTwo-week sitagliptin administration increased the plasma con-centration of GLP-1 in WKYs and SHRs (Figure S2A), as wellas GLP-1 and GLP-1R (Figure S2B and S2C) expressions inrenal arteries. Treatment with sitagliptin markedly augmentedacetylcholine-induced endothelium-dependent relaxation (EDR)in SHR renal arteries without affecting those in WKYs (Figure2A; pD2, 6.660.12 in SHRs versus 7.220.10 in WKYs and

Emax%: 25.81.7 in SHRs versus 76.35.7 in WKYs;P0.05). By contrast, endothelium-independent relaxationsto sodium nitroprusside were similar among all of the groups(Figure 2B). The phosphorylations of PKA C (Figure 2C),LKB1 (Figure 2D), AMPK (Figure 2E), and eNOS (Figure2F) were elevated in renal arteries from WKYs and SHRsafter sitagliptin treatment, which were reversed by SQ22536(100 mol/L, adenylyl cyclase inhibitor) and H89 (1 mol/L,PKA inhibitor; Figure 3A through 3D) or exendin 9-39 (100nmol/L; GLP-1R antagonist; Figure 4A through 4D). Theincreased phosphorylations of AMPK and eNOS (Figure 4Cand 4D) but not those of PKA C and LKB1 (Figure 4A and4B) were reversed by compound C (10 mol/L; AMPKinhibitor). SQ22536 (100 mol/L) and H89 (1 mol/L;Figure 3E), exendin 9-39 (100 nmol/L), compound C (10mol/L), and NG-nitro-L-arginine methyl ester (100 mol/L;NO synthase inhibitor; Figure 4E) also inhibited the im-proved EDR. Sitagliptin treatment in vivo increased cAMPlevels in SHR renal arteries, which were inhibited by exendin9-39 (100 nmol/L) and SQ22536 (100 mol/L) but not bycompound C (10 mol/L; Figure S3).

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Figure 3. Restoration of endothelial NO synthase (eNOS) activity by sitagliptin is mediated through cAMP and protein kinase A (PKA) inspontaneously hypertensive rat (SHR) renal arteries. Effects of 30-minute incubation with 100 mol/L of SQ22536 and 1 mol/L of H89on phosphorylations of PKA catalytic subunit (C; A), liver kinase B1 (LKB1; B), AMP-activated protein kinase (AMPK; C) and eNOS (D)in sitagliptin-treated SHR renal arteries. E, Effects of SQ22536 (100 mol/L) and H89 (1 mol/L) on endothelium-dependent relaxation(EDR) in renal arteries from sitagliptin-treated SHRs. Data are meanSEM. *P0.05 vs vehicle (Veh), #P0.05 vs sitagliptin. n4 forWestern blotting; n8 for relaxations.



Exendin 4 Improves Endothelium-DependentRelaxation in SHR Renal ArteriesGLP-1R agonist exendin 4 (10 nmol/L; 12 hours) increasedacetylcholine-induced EDR in SHR renal arteries, whichwere reversed by coincubation with SQ22536 (100 mol/L)and H89 (1 mol/L; Figure 5A) or compound C (10 mol/L)and NG-nitro-L-arginine methyl ester (100 mol/L; Figure5B) or exendin 9-39 (100 nmol/L) and GLP-1R antibody (2.5g/mL; Figure 5C). By contrast, exendin 4 treatment for 12hours had no effect on EDR in WKYs (Figure 5D).

Exendin 4 Increases AMPK and eNOSPhosphorylations and Stimulates NO Productionin SHR Aortic Endothelial CellsExendin 4 (10 nmol/L) stimulated NO production in primary SHRaortic endothelial cells, which was inhibited by pretreatment withexendin 9-39 (100 nmol/L), SQ22536 (100 mol/L), H89 (1mol/L), compound C (10 mol/L), or NG-nitro-L-arginine methylester (100 mol/L; Figure S4 and S5). Twelve-hour ex vivotreatment with either exendin 4 (10 nmol/L) or sitagliptin (10mol/L) increased the cGMP level in SHR renal arteries (FigureS6). Transient overexpression of AMPK2 by wild-type AMPK2

further increased AMPK and eNOS phosphorylations (Figure S7)and NO production (Figure S4D) in response to exendin 4 inendothelial cells, whereas suppression of the AMPK activity bydominant-negative AMPK2 (K45R mutated) inhibited such ef-fects. The level of AMPK2 increased significantly in SHRendothelial cells by expression of wild-type AMPK but notdominant-negative AMPK (Figure S8).

Sitagliptin Ameliorates Endothelial Dysfunction inRenal Arteries From Hypertensive PatientsEDRs were impaired in renal arteries from hypertensivepatients compared with those from normotensive patients,whereas exendin 4 (10 nmol/L; 12 hours) improved EDRsin renal arteries from hypertensive patients (Figure 6A).The reduced GLP-1R level (Figure 6B) and decreased phos-phorylations of PKA C, LKB1, AMPK, and eNOS (Figure6C through 6F) were elevated by exendin 4 in these arteries.

DiscussionThe present study demonstrated a functional importance ofGLP-1 and GLP-1R in the regulation of endothelial func-tion in SHR renal vasculature. The major novel findings

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Figure 4. Endothelial function improved by sitagliptin is glucagon-like peptide 1 receptor (GLP-1R) dependent in spontaneouslyhypertensive rat (SHR) renal arteries. A, Effects of exendin 9-39 (Ex9-39; 100 nmol/L) and compound C (CC; 10 mol/L) on phosphory-lations of protein kinase A catalytic subunit (PKA C; A), liver kinase B1 (LKB1; B), AMP-activated protein kinase (AMPK; C), and endo-thelial NO synthase (eNOS; D) in renal arteries from sitagliptin-treated SHRs. E, Effects of Ex9-39, CC, and NG-nitro-L-arginine methylester (l-NAME; 100 mol/L) on endothelium-dependent relaxation (EDR) in sitagliptin-treated SHRs renal arteries. Data are meanSEM.*P0.05 vs vehicle (Veh), #P0.05 vs sitagliptin. n6 for Western blotting; n8 for relaxations.



include the following: (1) treatment with sitagliptin in vivoand exendin 4 ex vivo improved endothelial function inSHR renal arteries via the sequential activation of thePKA/LKB1/AMPK/eNOS axis; (2) exendin 4 stimulatedNO production in SHR aortic endothelial cells and im-proved endothelial function in renal arteries from hyper-tensive patients; (3) GLP-1R expression was reduced inSHR renal arteries, which was upregulated by chronicsitagliptin treatment; and (4) 2-week oral administration ofsitagliptin to SHR lowered SBP and improved RBF with-out affecting glucose metabolism (Figure S9). Our studyreveals a protective role of GLP-1 and related agents inimproving endothelial function in hypertension throughthe activation of AMPK.

Clinical and experimental studies suggest that GLP-1 andits analogs confer cardiovascular protection through favor-able modulation of heart rate, blood pressure, and cardiachemodynamic responses.15,23,24 The present study shows thatin vivo sitagliptin or in vitro exendin 4 treatment improvedthe impaired EDR in SHR or hypertensive patients andstimulated NO production in primary SHR aortic endothelialcells, which is likely mediated by AMPK/eNOS activation.To further confirm the role of AMPK, the present studyshow that the suppression of AMPK2 activity by dominant-negative AMPK2 in SHR endothelial cells inhibited theAMPK/eNOS phosphorylations and NO production trig-gered by exendin 4, whereas increasing the AMPK activityby wild-type AMPK overexpression enhanced the stimula-tory effect of exendin 4. AMPK, which is one of the principalkinases responsible for phosphorylation and activation of eNOS,also mediates the vasoprotective effects of metformin and

berberine in endothelial cells.2528 In the present study, we used3 assay methods, including NG-nitro-L-arginine methyl estersensitive EDR in renal arteries, eNOS phosphorylation, and NOproduction, to support that NO bioavailability was increased bysitagliptin or exendin 4 treatment. NO levels in SHR endothelialcells were detected by the use of 4-amino-5-methylamino-2=,7=-difluorofluorescein fluorescence29 and the Total Nitric OxideAssay kit.30 After release, NO acts on vascular smooth musclecells to stimulate the activity of soluble guanylate cyclase, anenzyme that catalyzes the chemical conversion of GTP intocGMP.31 Therefore, we measured cGMP levels in SHR renalarteries that were elevated by 12-hour ex vivo incubation withexendin 4 or sitagliptin and thus confirmed the increased NOproduction (Figure S6). The present results suggest that GLP-1elevating agents could be a novel upstream regulator ofAMPK to preserve the NO bioavailability and endothelialfunction in hypertension.

GLP-1R is expressed in human coronary artery endothe-lial cells,32 as well as in the endothelium and smoothmuscle cells,23 which were shown to be downregulated byhyperglycemia, and this decrease likely contributed to theimpaired incretin effects in diabetes mellitus.33 The pres-ent study shows that GLP-1R expression was reduced inSHR renal arteries, whereas in vivo sitagliptin treatmentincreased the expression of GLP-1R, supporting thatDPP-4 inhibition restores the expression and function ofGLP-1/GLP-1R in SHR arteries. However, the mechanismof GLP-1R downregulation in hypertension needs to befurther elucidated.

The actions of GLP-1R are thought to involve cAMPproduction and PKA activation.23,34 Kieffer and Habener35

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Figure 5. Exendin 4 ameliorates endothelial dysfunction in spontaneously hypertensive rat (SHR) renal arteries. Reversal of theimproved endothelium-dependent relaxation (EDR) in exendin 4 (10 nmol/L, 12 hours)treated SHR renal arteries by cotreatment with(A) SQ22536 (100 mol/L) and H89 (1 mol/L), (B) compound C (CC; 10 mol/L) and NG-nitro-L-arginine methyl ester (l-NAME; 100mol/L, 30 minutes), or (C) by exendin 9-39 (Ex9-39; 100 nmol/L) and glucagon-like peptide 1 receptor (GLP-1R) antibody (GLP-1R Ab;2.5 g/mL, 2 hours). Control and exendin 4 groups are similar in A through C. D, Exendin 4 (10 nmol/L, 12 hours) had no effect on EDRin renal arteries from Wistar-Kyoto rats (WKYs). Data are meanSEM. *P0.05 vs control. #P0.05 vs exendin 4. n4 for WKYs; n6for SHRs.



suggested the role of the GLP-1R and cAMP in the actions ofGLP-1 on vascular endothelium. Moreover, PKA stimulatesLKB1 for AMPK activation in hepatocytes.36 The presentstudy demonstrated that sitagliptin stimulated the activationof LKB1/AMPK subsequent to cAMP/PKA signaling onactivation of GLP-1R (Figure S10). Finally, the in vivo effectof sitagliptin was assessed in SHRs by measuring RBF andblood pressure. Chronic GLP-1 treatment lowers blood pres-sure in patients with type 2 diabetes mellitus,37 and exendin4 also exerts an antihypertensive effect in salt-sensitivehypertensive mice.13,17 Another study suggests that DPP-4inhibition by sitagliptin attenuates blood pressure elevation inyoung SHRs by diminishing proximal tubule sodium reab-sorption.38 In the present study, we observe that 2-weeksitagliptin treatment reduced SBP in adult SHRs. The resto-ration of AMPK/eNOS in SHR arteries after sitagliptintreatment might also contribute in part to blood pressurereduction.39,40 Renal function is of importance in controllingblood pressure in the development of essential hypertension,3and RBF is a parameter of renal function.40 RBF is reducedin renovascular beds in essential hypertension, which isattributed to arteriolar constriction.41 SHRs are known tohave higher mean arterial pressure and renal vascular resis-tance than WKYs under baseline conditions. Moreover, NOsynthase inhibition induced hypertension42 and caused morereduction of RBF in SHRs,43 suggesting that RBF is closelyrelated to the NO availability in renal circulation. The present

study showed that RBF was reduced in SHRs compared withWKYs, and 2-week sitagliptin treatment increased RBF inSHRs, which may in part contribute to the blood pressurelowering effect of sitagliptin.

PerspectivesWe demonstrate that DPP-4 inhibition by sitagliptin pre-serves vascular GLP-1/GLP-1R function in SHRs, a geneticmodel of hypertension. GLP-1induced AMPK/eNOS acti-vation restores endothelium-dependent relaxation and RBF,thus helping to reduce SBP in SHRs. Like sitagliptin, theGLP-1R agonist exendin 4 is also effective in augmentingendothelial function in hypertensive rats and patients, therebyelucidating the mechanism underlying the vascular benefitsof GLP-1 and related agents. Taken together, the novelfindings of the present study highlight the prospect for the useof GLP-1elevating agents and GLP-1R agonists againstvascular dysfunction in hypertension.

AcknowledgmentWe thank Merck Research Laboratories for the generous gift ofsitagliptin for the present study.

Sources of FundingThis study is supported by the National Basic Research Program ofChina (2012CB517805), research grants (466110 and HKU4/CRF10) from the Research Grants Council of Hong Kong, andChinese University of Hong Kong Focused Investment Scheme B.

0.0

0.5

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NT

ACh (log mol/L)

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axat

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FNT C E

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HT

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Figure 6. Exendin 4 ameliorates endothelial dysfunction in renal arteries from hypertensive patients. A, Endothelium-dependentrelaxation (EDR) in the renal arteries from patients. Expression of glucagon like peptide 1 receptor (GLP-1R; B) and phosphoryla-tions of protein kinase A catalytic subunit (PKA C; C), liver kinase B1 (LKB1; D), AMP-activated protein kinase (AMPK; E), andendothelial NO synthase (eNOS; F) in human renal arteries. Data are meanSEM. *P0.05 vs NT (normotensive patients).#P0.05 vs HT (hypertensive patients). C indicates control; E, exendin 4. n4 for Western blotting; n4 for HT relaxations; n6for NT relaxations.



DisclosuresNone.

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Novelty and Significance

What Is New? Treatment with the dipeptidyl peptidase 4 inhibitor sitagliptin and GLP-1

receptor agonist exendin 4 improves endothelial function. Sitagliptin treatment lowers SBP and enhances RBF in spontaneously

hypertensive rats. Sitagliptin treatment increases vascular GLP-1 receptor expression. Exendin 4 stimulates NO production and improves endothelial function in

hypertensive patients.

What Is Relevant? The novel findings of the present study highlight the prospect for the use

of GLP-1--elevating agents and GLP-1 receptor agonists against vascu-lar dysfunction in hypertension.

Summary

The upregulation of GLP-1 and related agents preserves endothe-lial function in hypertension by restoring NO bioavailability.



Liu et al., 2012, Supplemental Materials

1

Dipeptidyl Peptidase-4 Inhibitor Sitagliptin Protects Endothelial Function

in Hypertension through GLP-1 Dependent Mechanism

Limei Liu1,2, Jian Liu1, Wing Tak Wong1, Xiao Yu Tian1, Chi Wai Lau1, Yi-Xiang Wang3, Gang

Xu4, Yunfei Pu5, Zhiming Zhu5, Aimin Xu6,7, Karen SL Lam6, Zhen Yu Chen8, Chi Fai Ng9, Xiaoqiang Yao1, Yu Huang1

1Institute of Vascular Medicine, Li Ka Shing Institute of Health Sciences, and School of Biomedical Sciences, Chinese University of Hong Kong, Hong Kong, China; 2Department of Physiology and

Pathophysiology, Peking University Health Science Center, Peking, China; 3Department of Imaging and Interventional Radiology , Chinese University of Hong Kong, Hong Kong, China; 4Department of Medicine and Therapeutics, Chinese University of Hong Kong, Hong Kong, China; 5Department of

Hypertension and Endocrinology, Daping Hospital, Third Military Medical University, China; Departments of 6Medicine and 7Pharmacology and Pharmacy, University of Hong Kong, Hong Kong, China; 8School of Life Sciences and 9Department of Surgery, Chinese University of Hong Kong, Hong

Kong, China

Correspondence: Yu Huang ([email protected]) or Limei Liu ([email protected])

Supplemental Materials Expanded Materials and Methods Animals Male spontaneously hypertensive rats (SHRs) and Wistar-Kyoto rats (WKYs) were supplied by the Chinese University of Hong Kong (CUHK) Laboratory Animal Service Center. This investigation was approved by the CUHK Animal Experimentation Ethics Committee and conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1996). SHRs (32~40 weeks old) and WKYs (32~40 weeks old) received sitagliptin (10 mg/kg/day by oral gavage) or vehicle for 2 weeks.

Blood Pressure Measurement SHRs were surgically implanted with telemetric transmitters (TL11M2-C50-PXT, Data Sciences International, Minnesota, USA). The catheter of the implant was placed into the distal portion of the descending aorta. Rats were allowed to recover from surgery for 7 days, and then 24h ambulatory systolic blood pressures were measured by telemetry in conscious, unrestrained rats. Data were collected for 20 seconds every 20 min and used the 24-hour mean values for analysis. The ambulatory arterial pressures were measured at Day 0 and Day 14 after sitagliptin treatment in SHRs and WKYs. For direct catheter measurement, vehicle and sitagliptin-treated WKY and SHR were anesthetized.

Systolic blood pressure was measured by inserting a heparinized saline-filled PE-50 catheter into the left common carotid artery after an initial 15 min equilibration period.1 In addition, systolic blood pressure was also measured by the tail-cuff method before and after sitagliptin treatment. Blood pressure was calculated from the average of 5 successive recordings. Measurement of Renal Blood Flow by Magnetic Resonance Image (MRI) Acquisition Procedure MRI studies were performed using a 3T clinical whole-body imaging system (Achieva, Philips Healthcare, Best, Netherlands). MRI contrast agent was gadolinium-tetraazacyclododecanetetraacetic acid (Gd-DOTA) (Guerbet Group, Roissy CDG cedex, France). After anesthesia, rats were positioned supinely. The MRI acquisition of the rat urinary system included high resolution T2 weighted axial plane anatomical examination, high resolution T1 weighted coronal plane anatomical examination, and dynamic contrast enhanced examination in coronal plane. Axial anatomical examinations were acquired with the following parameters: multiple slice turbo spine echo sequence, repetition time (TR)/ time to echo (TE)/flip angle= 2359 ms/120 ms/90, field of view = 60 mm81 mm30 mm, the acquisition voxel size was 0.41 mm0.41 mm1.50 mm, and the reconstructed voxel size was 0.17 mm0.17 mm1.5 mm. Coronal anatomical examinations were acquired with the following parameters: three-dimensional (3D)



2

gradient echo sequence with fat suppression, TR/TE/flip angle= 4.4 ms/2.2 ms/10, field of view = 80 mm 80 mm18 mm, the acquisition voxel size was 0.50 mm0.50 mm1.00 mm and the reconstructed voxel size was 0.28 mm0.28 mm0.50 mm. The contrast-enhanced examinations were acquired with the following parameters: 3D gradient echo sequence, TR/TE/flip angle= 6.8 ms/2.3 ms/35, field of view = 80 mm80 mm12 mm, the acquisition voxel size was 0.61 mm0.75 mm3.00 mm and the reconstructed voxel size was 0.31 mm0.31 mm1.5 mm. MRI contrast agent was gadolinium-tetraazacyclododecanetetraacetic acid (Gd-DOTA) (Guerbet Group, Roissy CDG cedex, France). A dose of 0.075 mmol/kg was injected through tail vein as a rapid bolus in less than 1 sec after initial baseline 10 acquisitions and followed by a flush of 0.5 mL normal saline. Dynamic scan was stopped when the contrast agent was excreted and clearly visible in the bilateral ureters. MRI Analysis The reconstructed MR images were transferred to a radiological workstation (Extended Workspace, Philips, Best, Netherlands) for off-line analysis. Anatomical images were read by a radiologist with animal research experiences. For analysis of dynamic data, regions of interest (ROIs) were manually drawn over left and right kidneys. The ROIs of the renal cortex were drawn in all rats. These ROIs were used on the perfusion-weighted data to generate time signal intensity curves. Intrarenal Artery Preparation Rats were sacrificed by CO2 suffocation and intrarenal arteries were removed and placed in ice-cold Krebs solution (mmol/L): 119 NaCl, 4.7 KCl, 2.5 CaCl2, 1 MgCl2, 25 NaHCO3, 1.2 KH2PO4, and 11 D-glucose. Arteries were cleaned of adhering tissue and cut into ring segments of 2 mm in length. Arteries from SHR were incubated for 12 hours in Dulbeccos Modified Eagles Media (DMEM, Gibco, Grand Island, NY, USA) culture media with 10% fetal bovine serum (FBS, Gibco), 100 IU penicillin and 100 g/mL streptomycin with or without sitagliptin or exendin-4. Rings were suspended in myograph (Danish Myo Technology, Aarhus, Denmark) for recording of changes in isometric tension.2, 3 Briefly, two tungsten wires (40 m in diameter) were inserted through the lumen and fixed to jaws of organ chamber. The organ chamber was filled with 5 mL Krebs solution and gassed by 95% O2-5% CO2 at 37C (pH ~7.4). Each ring was stretched to 2.5 mN, an optimal tension, and then allowed to stabilize for 90 min before the start of each experiment.2

Functional Studies Each ring was initially contracted by 60 mmol/L KCl. Endothelium-dependent relaxation (EDR) to

acetylcholine (ACh, 0.003 to 10 mol/L) while endothelium-independent relaxation to sodium nitroprusside (SNP, 0.001 to 10 mol/L) were examined in arteries pre-contracted with phenylephrine (1 mol/L). In the first set of experiments, SHR renal arteries were incubated with exendin-4 (10 nmol/L, GLP-1 receptor agonist) for 12 hours before vasoreactivity study on wire myograph. In some experiments, GLP-1 receptor antibody (2.5 g/mL) was added 2 hours before incubation with exendin-4 or incubation with SQ22536 (100 mol/L, adenylate cyclase inhibitor), H89 (1 mol/L, PKA inhibitor), exendin 9-39 (100 nmol/L, GLP-1 receptor antagonist) and compound C (10 mol/L, AMPK inhibitor) along with exendin-4. Some arterial rings were subjected to 30-min exposure to L-NAME (100 mol/L, nitric oxide synthase inhibitor) and then endothelium-dependent relaxations in response to cumulative additions of ACh were measured. The second series of experiments examined the impact of oral treatment with sitagliptin on endothelial function in SHRs. The relaxations to ACh in renal arteries from sitagliptin-treated SHRs were studied in control and in the presence of each of the following inhibitors (30-min incubation): SQ22536 (100 mol/L), H89 (1 mol/L), exendin 9-39 (100 nmol/L),4 compound C (10 mol/L),5 or L-NAME (100 mol/L). Measurement of GLP-1 in Plasma Plasma was kept from vehicle and sitagliptin-treated WKYs and SHRs. GLP-1 levels in plasma were assayed by Glucagon-Like Peptide-1 (Active) ELISA kit (Linco Research) according to the manufacturers instructions. cAMP Levels in Renal Arteries Renal arteries from sitagliptin-treated SHR were cultured with or without inhibitors and were prepared according to the manufacturers instructions. cAMP levels were assayed by Direct cAMP ELISA Kit (Enzo Life Sciences, Farmingdale, NY, USA). Primary Culture of Rat Aortic Endothelial Cells Aortas of SHR were dissected in sterilized phosphate buffered saline (PBS) under a stereoscopic microscope. After digestion by 0.2% collagenase for 15 minutes at 37C, RPMI-1640 (Gibco) was added and endothelial cells were then collected by centrifugation at 1000 rpm for 5 minutes. Thereafter, the pellet was gently re-suspended in RPMI-1640 supplemented with 10% FBS and cultured in a 75-cm2 cell culture flask. To remove other cell types, the medium was changed after 1-hour incubation, then maintained until 70% confluence before use. Transfection Condition SHR aortic endothelial cells were transfected with either a wild type AMPK2 plasmid (WT-AMPK), a



3

dominant negative AMPK construct K45R (DN-AMPK), or control vector by electroporation using Nucleofector II machine (Amaxa/Lonza, Walkersville, MD, USA) according to the manufacturers instruction. About 70% of endothelial cells were successfully transfected as indicated by control transfection using a GFP-expressing pCAGGS vector. Western Blot Analysis Isolated renal arteries or SHR aortic endothelial cells were homogenized in RIPA lysis buffer that contained 1 g/mL leupeptin, 5 g/mL aprotinin, 100 g/mL PMSF, 1 mmol/L sodium orthovanadate, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L sodium fluoride, and 2 g/mL -glycerolphosphate, and centrifuged at 20,000 g for 20 min at 4C. Protein lysates (25 g for arteries, 10 g for cells) were separated by electrophoresis and transferred onto PVDF membrane. Blots were blocked with 1% bovine serum albumin or 5% non-fat milk for 1 hour and incubated overnight at 4C with antibodies against phospho-PKA C (catalytic subunit, Thr197), phospho-LKB1 (Ser334), phospho-eNOS (Ser1177), phospho-AMPK (Thr172), PKA C, LKB1, eNOS, AMPK and AMPK2, GLP-1 receptor and against mouse GLP-1 and GAPDH. After washing, blots were incubated with HRP-conjugated swine anti-rabbit or anti-mouse IgG. Immunoreactive bands were visualized by chemiluminescence and exposed to Kodak Image Station 440 for densitometric analysis. Nitric Oxide (NO) Measurement Endothelial cells seeded on glass coverslips were loaded with 1 mol/L DAF-FM diacetate (Molecular Probes, Eugene, OR, USA) at room temperature for 10 minutes and placed in a designed chamber for fluorescence imaging. Intracellular NO production was monitored using a fluorescent NO indicator DAF-FM diacetate as described.6 DAF-FM diacetate is cell-permeant and passively diffuses across cellular membrane. The fluorescence quantum yield of DAF-FM is ~0.005, but increases ~160 fold to ~0.81, after reacting with NO, which was measured by a confocal scanning unit (FV1000, Olympus, Tokyo, Japan) at excitation 488 nm and an emission filter of 505-525 nm. Changes in [NO]i were displayed as a ratio of fluorescence relative to the intensity (F1/F0), and analyzed by the Fluoview software (Olympus). Total NO Production in Endothelial Cells SHR endothelial cells were incubated in the presence of exendin-4 (10 nmol/L, 30min) with or without inhibitors. Total NO production in SHR endothelial cells was determined by measuring the concentration of nitrate and nitrite, a stable metabolite of NO, by the Total Nitric Oxide Assay Kit (Beyotime Biotechnology) according to the manufacturers instructions. cGMP Levels in Renal Arteries SHR renal arteries were cultured with sitagliptin (10 mol/L) or exendin-4 (10 nmol/L) for 12 hours. The

tissue were then frozen and stored at -80 C until assay. The levels of cyclic GMP were measured by direct cGMP ELISA Kit (Enzo Life Sciences, Farmingdale, NY, USA) according to the manufacturers instruction. The result was expressed as cyclic GMP production in pmol per mg protein. Human Artery Specimen The present study was approved by the Joint Chinese University of Hong Kong-New Territories East Cluster Clinical Research Ethics Committee. Human renal arteries were obtained after informed consent from normotensive and hypertensive patients undergoing nephrectomy at ages between 50-80 years old. The indications for surgery included tumor (4 in normotensive patients and 3 in hypertensive patients) and poorly functioning kidney (2 in normotensive patients and 1 in hypertensive patients). History of hypertension was defined as having persistent elevated blood pressure, systolic blood pressure of >140 mm Hg, or diastolic blood pressure of >90 mm Hg and requiring medical therapy. Materials and Drugs Anti-phospho-eNOS (Ser1177), anti-eNOS, anti-GLP-1 receptor and anti-GLP-1 antibodies were obtained from Abcam (Cambridge, MA). Anti-phospho-PKA C (Thr197), phosphor-LKB1 (Ser334), phospho-AMPK (Thr172), anti-AMPK, anti-PKA C, anti-LKB1 and anti-AMPK2 antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against GAPDH were obtained from Ambion (Austin, TX, USA). HRP-conjugated swine anti-rabbit or anti-mouse IgG were from DakoCytomation (Carpinteria, CA, USA). Immobilon-P polyvinylidene difluoride (PVDF) membrane was from Millipore (Billerica, MA, USA) and chemiluminescence (ECL reagents) was obtained from Amersham Pharmacia. Phenylephrine, acetylcholine, L-NAME, sodium nitroprusside, H89, compound C, exendin-4, exendin 9-39 were purchased from Sigma-Aldrich Chemical (St Louis, MO, USA). SQ22536 was from Calbiochem (San Diego, CA, USA). The cell culture media and DAF-FM diacetate were from Invitrogen (Carlsbad, CA, USA). Sitagliptin was a kind gift from Merck Research Laboratories (Rahway, NJ, USA). SQ22536 and compound C were dissolved in DMSO and other drugs in distilled water. DMSO (0.1% v/v) did not modify agonist-induced responses. Data Analysis Results represent meansSEM from different animals. Concentration-response curves were analyzed by non-linear curve fitting using GraphPad Prism software (Version 4.0, San Diego, CA, USA). The negative logarithm of the dilator concentration that produced half of the maximum effect (pD2) and



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the maximum relaxation (Emax%) were calculated. The protein expression was quantified by densitometer (FluorChem, Alpha Innotech, San Leandro, CA, USA), and analyzed by Quantity One software (Bio-Rad). Statistical significance was determined by two-tailed Students t-test or one-way ANOVA followed by the Bonferroni post-hoc test when more than two treatments were compared. p


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receptors dilates cortical efferent arterioles in mouse. Kidney Int. 2009;75:793-799.

11. Guan Z, Pollock JS, Cook AK, Hobbs JL, Inscho EW. Effect of epithelial sodium channel blockade

on the myogenic response of rat juxtamedullary afferent arterioles. Hypertension. 2009;54:1062-1069.



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Supplemental Figures A B

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Figure S1. Systolic blood pressure (SBP) of rats. SBP were measured by direct measurement of SBP with a direct catheter in anesthetized rats (A) and the tail-cuff method (B). Two-week oral administration with sitagliptin (10 mg/kg/day) decreased SBP of SHRs without affecting the SBP of WKYs. (C) The ambulatory systolic (SBP), diastolic (DBP), and mean arterial (MABP) pressures and heart rates (HR) were shown in sitagliptin-treated SHRs and WKYs compared to the vehicle-treated controls. Data are meansSEM. *p


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Figure S2. Levels of GLP-1 and GLP-1 receptor in rats. Two-week oral administration of sitagliptin (10 mg/kg/day) increased the plasma concentration of GLP-1 (A), and GLP-1 (B) and GLP-1 receptor (C) expressions in renal arteries from WKY and SHRs. Data are meansSEM. *p


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Figure S4. Exendin-4 stimulates NO production in SHR aortic endothelial cells. Inhibitory effects of exendin 9-39 (Ex9-39, 100 nmol/L) (A), SQ22536 (100 mol/L) and H89 (1 mol/L) (B), compound C (CC, 10 mol/L) and L-NAME (100 mol/L) (C) on NO production stimulated by exendin-4 (ex4). (D) Over-expression of AMPK2 (WT) further elevated NO production stimulated by exendin-4 and these effects were inhibited by suppression of AMPK activity through expression of DN-AMPK (DN). (E) Images showing NO production under various treatments. Data are meansSEM. *p


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Figure S5. Total NO production in SHR aortic endothelial cells. Total NO production in SHR endothelial cells was determined by measuring the concentration of nitrate and nitrite, a stable metabolite of NO, by modified Griess reaction method. Exendin-4 (10 nmol/L, 30min) increased NO production, which was inhibited by exendin 9-39 (100 nmol/L), SQ22536 (100 mol/L), H89 (1 mol/L), or compound C (10 mol/L). Data are meansSEM. *p


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Figure S7. Exendin-4 increases AMPK and eNOS phosphorylations in SHR aortic endothelial cells. Overexpression of AMPK2 (WT-AMPK, WT) further increased phosphorylations of AMPK (A) and eNOS (B) stimulated by exendin-4 (10 nmol/L, 12 hours) in SHR endothelial cells, while expression of DN-AMPK (DN) inhibited these effects. Data are meansSEM. *p


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Figure S9. Insulin tolerance test (ITT) of rats. There was no difference in ITT between WKY and SHR at the age used in the present study. Sitagliptin had no effect on ITT in SHR. Data are meansSEM. n=4.

Figure S10. The proposed cellular mechanism for the protective effect of sitagliptin against endothelial dysfunction in hypertension. The bioavailability of NO decreased in hypertension. Sitagliptin enhances phosphorylation of eNOS at Ser1177 via AMPK phosphorylation at Thr172 through the activation of GLP-1R/cAMP/PKA/LKB1 cascade, leading to elevated NO level and thus improves endothelial function in hypertension.

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