Yao and Yu Huang

Yunfei 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 Mechanism Like Peptide 1 Through 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 75231 Hypertension
doi: 10.1161/HYPERTENSIONAHA.112.195115
2012;60:833-841; originally published online August 6, 2012; Hypertension.
http://hyper.ahajournals.org/content/60/3/833
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Dipeptidyl Peptidase 4 Inhibitor Sitagliptin Protects
Endothelial Function in Hypertension Through a
GlucagonLike 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 like
peptide 1 (GLP-1), which is used for the treatment of type 2 diabetes mellitus. However, little is known about the role
of GLP-1 in hypertension. This study investigated whether the activation of GLP-1 signaling protects endothelial
function 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 spontaneously
hypertensive 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 NO
synthase in spontaneously hypertensive rat renal arteries. Inhibition of GLP-1 receptor, adenylyl cyclase, protein kinase
A, AMP-activated protein kinase-, or NO synthase reversed the protective effects of sitagliptin. We also demonstrate
that GLP-1 receptor agonist exendin 4 in vitro treatment had similar vasoprotective effects in spontaneously
hypertensive 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 the
critical role of AMP-activated protein kinase- in mediating the effect of GLP-1 in endothelial cells. Ex vivo exendin
4 treatment also improved endothelial function of renal arteries from hypertensive patients. Our results elucidate that
upregulation 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
H
ypertension is caused by pathological changes in renal
and vascular structure and function involved in blood
pressure regulation.
1
Hypertension can cause renal damage if
it is not properly controlled.
2
The impaired vasodilator
response is a risk factor for renal function loss in patients with
essential hypertension.
3
Persistent hypertension alters func-
tional characteristics of vascular endothelial cells and is
associated with impaired vasodilatory function.
4
Diminished
production and function of endothelium-derived NO leads to
endothelial dysfunction,
5
a crucial initial step culminating in
vascular events in hypertension.
Dipeptidyl peptidase 4 (DPP-4), also known as CD26, is a
ubiquitous enzyme detectable in the endothelium.
6
Glucagon-
like peptide 1 (GLP-1) produced by L-type cells in the
intestine, is a substrate for DPP-4.
7
GLP-1 improves glucose
use in patients with type 2 diabetes mellitus by increasing
insulin secretion and inhibiting glucagon secretion.
8,9
Sita-
gliptin, a highly selective DPP-4 inhibitor,
10
inhibits the
inactivation and degradation of GLP-1,
11
which is used for
the treatment of type 2 diabetes mellitus as monotherapy
or in combination with other antiglycemic agents, such as
metformin.
12
The effect of GLP-1 on blood pressure has been reported in
both animal and human hypertension.
13,14
Treatment with
GLP-1 receptor (GLP-1R) agonists leads to a transient blood
pressure 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 yu-huang@cuhk.edu.hk,
and Limei Liu, Department of Physiology and Pathophysiology, Peking University Health Science Center, Beijing, China. E-mail liu_limei@126.com
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, 2012 http://hyper.ahajournals.org/ Downloaded from
outflow.
15
However, continuous infusion of GLP-1 produces
a small but insignificant reduction of blood pressure in
patients with type 2 diabetes mellitus.
16
Moreover, chronic
administration of recombinant GLP-1 prevents the develop-
ment of hypertension and improves endothelial function in
Dahl salt-sensitive rats.
17
By contrast, the effect of DPP-4
inhibition on blood pressure is largely unknown, although
limited studies show that DPP-4 inhibition by sitagliptin
produces a small blood pressurelowering effect in nondia-
betic patients with hypertension on stable antihypertensive
therapy.
18
The present study investigated whether DPP-4
inhibition could ameliorate endothelial dysfunction in renal
arteries from hypertensive animals and patients, as well as
possible signaling mechanisms involved.
Materials and Methods
A supplemental Methods section can be found in the online-only
Data Supplement.
0
1000
2000
3000
4000
5000
WKY SHR
Sitagliptin - + - +
*
#
A
U
C

(
S
y
s
t
o
l
i
c

B
P

X

2
4

h
)
0 6 12 18 24
100
150
200
250
SHR Sitagliptin
WKY Sitagliptin
SHR Vehicle
WKY Vehicle
Day 14
Time (hour)
S
B
P

(
m
m
H
g
)
0 6 12 18 24
100
150
200
250
Day 0
Time (hour)
S
B
P

(
m
m
H
g
)
100
120
140
160
180
200
WKY SHR
Sitagliptin
- + - +
*
#
A
v
e
r
a
g
e
d

S
B
P

(
m
m
H
g
)
W
K
Y

V
e
h
i
c
l
e
S
H
R

V
e
h
i
c
l
e
S
H
R

S
i
t
a
g
l
i
p
t
i
n
0
50
100
150
M
a
x
i
m
u
m

e
n
h
a
n
c
e
m
e
n
t
(
A
r
e
a

u
n
d
e
r

c
u
r
v
e
)

1
0
0
0
0 50 100
0
1000
2000
1 - WKY Vehicle
2 - SHR Sitagliptin
3 - SHR Vehicle
1
2
3
Time (Sec)
M
a
x
i
m
u
m

e
n
h
a
n
c
e
m
e
n
t
(
R
e
n
a
l

b
l
o
o
d

f
l
o
w
)
#
*
SHR
Sitagliptin
SHR
Vehicle
WKY
Vehicle
*
#
A
B
Figure 1. Sitagliptin lowers blood pressure and increases renal blood ow in spontaneously hypertensive rats (SHRs). A, Systolic blood
pressure (SBP) in vehicle and sitagliptin-treated Wistar-Kyoto rats (WKYs) and SHRs. B, Renal blood ow in WKY vehicle (1) rats or in
vehicle (3) and sitagliptin-treated SHRs (2). Data are meanSEM. *P0.05 vs WKY vehicle, #P0.05 vs SHR vehicle. n4.
834 Hypertension September 2012
at Chinese University of Hong Kong on August 30, 2012 http://hyper.ahajournals.org/ Downloaded from
Measurement and Analysis of Renal Blood Flow
by MRI Acquisition Procedure
MRI studies were performed using a 3T clinical whole-body imaging
system (Achieva, Philips Healthcare, Best, the Netherlands).
19
Renal Artery Preparation and Functional Studies
Rats were euthanized by CO
2
suffocation, and renal interlobar
arteries were removed and placed in ice-cold Krebs solution. Arteries
were prepared, and changes of isometric tension were recorded.
20,21
Western Blot Analysis
Protein expression levels of GLP-1, GLP-1R, phospho-PKA C
(protein kinase A catalytic subunit, Thr
197
), phospho-LKB1 (liver
kinase B1, Ser
334
), phospho-endothelial NO synthase (eNOS;
Ser
1177
), phosphoAMP-activated protein kinase (AMPK; Thr
172
),
PKA C, LKB1, eNOS, AMPK, and AMPK2 were detected by
Western blotting.
NO Measurement
Intracellular NO production was monitored using a fluorescent NO indica-
tor 4-amino-5-methylamino-2=,7=-difluorofluorescein diacetate
22
and the
Total Nitric Oxide Assay kit.
Data Analysis
Results represent meanSEM from different rats or patients. Statis-
tical significance was determined by 2-tailed Student t test or 1-way
ANOVA followed by the Bonferroni post hoc test when 2
treatments were compared. P values 0.05 indicate statistically
significant difference.
Results
Sitagliptin Treatment Lowers Blood Pressure and
Increases Renal Blood Flow in Spontaneously
Hypertensive Rats
Ambulatory 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 SHRs
compared with vehicle-treated SHRs (averaged SBP, 1608
versus 1805 mm Hg; n4 each group), whereas sitaglitin
treatment did not alter SBPs in Wistar-Kyoto rats (WKYs;
averaged SBP, 1204 versus 1194 mm Hg; n4 each
group; Figure 1A). This is further confirmed by the direct
-9 -8 -7 -6 -5
0
50
100
WKY SHR
Vehicle
Sitagliptin
ACh (log mol/L)
R
e
l
a
x
a
t
i
o
n
(
%

P
h
e

t
o
n
e
)
#
*
-9 -8 -7 -6 -5
0
50
100 WKY SHR
Vehicle
Sitagliptin
SNP (log mol/L)
R
e
l
a
x
a
t
i
o
n
(
%

P
h
e

t
o
n
e
)
0
1
2
p
P
K
A

C
/
P
K
A

C

(
C
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m
p
a
r
e
d

t
o

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Y

v
e
h
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c
l
e
)
0
1
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p
L
K
B
1
/
L
K
B
1
(
C
o
m
p
a
r
e
d

t
o

W
K
Y

v
e
h
i
c
l
e
)
p-LKB1(Ser
334
)
LKB1
GAPDH
C D
#
*
Sitagliptin - + - +
WKY SHR
p-PKA C(Thr
197
)
PKA C
GAPDH
#
*
Sitagliptin - + - +
WKY SHR
p-eNOS(Ser
1177
)
eNOS
GAPDH
E F
0
1
2
p
-
A
M
P
K
/
A
M
P
K

(
C
o
m
p
a
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e
d

t
o

W
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Y

v
e
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i
c
l
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)
0
1
2
p
-
e
N
O
S
/
e
N
O
S
(
C
o
m
p
a
r
e
d

t
o

W
K
Y

v
e
h
i
c
l
e
)
#
*
Sitagliptin - + - +
WKY SHR
p-AMPK(Thr
172
)
AMPK
GAPDH
#
*
*
Sitagliptin - + - +
WKY SHR
A B
Figure 2. Endothelial function improved
by sitagliptin is via restoring endothelial
NO synthase (eNOS) activity in sponta-
neously hypertensive rat (SHR) renal
arteries. A, Endothelium-dependent
relaxation (EDR) in renal arteries from
vehicle and sitagliptin-treated Wistar-
Kyoto rats (WKYs) and SHRs. B,
Endothelium-independent relaxations to
sodium nitroprusside (SNP) in renal
arteries from vehicle and sitagliptin-
treated rats. Phosphorylations of protein
kinase A catalytic subunit (PKA C) at
Thr
197
(C), LKB1 (liver kinase B1) at
Ser
334
(D), AMP-activated protein kinase
(AMPK) at Thr
172
(E), and eNOS at
Ser
1177
(F) in renal arteries from vehicle
and sitagliptin-treated WKYs and SHRs.
Data are meanSEM. *P0.05 vs WKY
vehicle, #P0.05 vs SHR vehicle. n10
for acetylcholine (ACh)-induced relax-
ations; n4 for SNP induced relaxations;
n6 for Western blotting.
Liu et al DPP-4 Inhibition Restores Endothelial Function 835
at Chinese University of Hong Kong on August 30, 2012 http://hyper.ahajournals.org/ Downloaded from
measurement of SBP with a direct catheter in anesthetized
rats (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 arterial
blood pressure or diastolic blood pressure in all groups of
rats. It is noted that the effect of sitagliptin on SBP in
SHRs was a step decrease that occurred between days 3
and 4 and that there was no diurnal rhythm in control and
sitagliptin-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 SHR
Renal Arteries
Two-week sitagliptin administration increased the plasma con-
centration of GLP-1 in WKYs and SHRs (Figure S2A), as well
as GLP-1 and GLP-1R (Figure S2B and S2C) expressions in
renal arteries. Treatment with sitagliptin markedly augmented
acetylcholine-induced endothelium-dependent relaxation (EDR)
in SHR renal arteries without affecting those in WKYs (Figure
2A; pD
2
, 6.660.12 in SHRs versus 7.220.10 in WKYs and
E
max
%: 25.81.7 in SHRs versus 76.35.7 in WKYs;
P0.05). By contrast, endothelium-independent relaxations
to 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 (Figure
2F) were elevated in renal arteries from WKYs and SHRs
after 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 (100
nmol/L; GLP-1R antagonist; Figure 4A through 4D). The
increased phosphorylations of AMPK and eNOS (Figure 4C
and 4D) but not those of PKA C and LKB1 (Figure 4A and
4B) were reversed by compound C (10 mol/L; AMPK
inhibitor). SQ22536 (100 mol/L) and H89 (1 mol/L;
Figure 3E), exendin 9-39 (100 nmol/L), compound C (10
mol/L), and N
G
-nitro-L-arginine methyl ester (100 mol/L;
NO synthase inhibitor; Figure 4E) also inhibited the im-
proved EDR. Sitagliptin treatment in vivo increased cAMP
levels in SHR renal arteries, which were inhibited by exendin
9-39 (100 nmol/L) and SQ22536 (100 mol/L) but not by
compound C (10 mol/L; Figure S3).
0
1
2
p
P
K
A

C
/
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K
A

C

(
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p
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)
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(
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e
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t
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v
e
h
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c
l
e
l
)
# #
*
#
#
*
p-AMPK(Thr
172
)
AMPK
GAPDH
p-eNOS(Ser
1177
)
eNOS
GAPDH
Veh Sitagliptin
Veh Sitagliptin
- - + -
- - - +
SQ22536
H89
- - + -
- - - +
SQ22536
H89
C
# #
*
#
#
*
p-PKA C(Thr
197
)
PKA C
GAPDH
p-LKB1(Ser
334
)
LKB1
GAPDH
Veh Sitagliptin Veh Sitagliptin
SHR SHR
- - + -
- - - +
SQ22536
H89
- - + -
- - - +
SQ22536
H89
A B
-9 -8 -7 -6 -5
0
50
100
Vehicle
Sitagliptin
H89+sitagliptin
SQ22536+sitagliptin
ACh (log mol/L)
R
e
l
a
x
a
t
i
o
n
(
%

P
h
e

t
o
n
e
)
#
#
*
E
SHR
SHR
D
Figure 3. Restoration of endothelial NO synthase (eNOS) activity by sitagliptin is mediated through cAMP and protein kinase A (PKA) in
spontaneously hypertensive rat (SHR) renal arteries. Effects of 30-minute incubation with 100 mol/L of SQ22536 and 1 mol/L of H89
on 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 for
Western blotting; n8 for relaxations.
836 Hypertension September 2012
at Chinese University of Hong Kong on August 30, 2012 http://hyper.ahajournals.org/ Downloaded from
Exendin 4 Improves Endothelium-Dependent
Relaxation in SHR Renal Arteries
GLP-1R agonist exendin 4 (10 nmol/L; 12 hours) increased
acetylcholine-induced EDR in SHR renal arteries, which
were reversed by coincubation with SQ22536 (100 mol/L)
and H89 (1 mol/L; Figure 5A) or compound C (10 mol/L)
and N
G
-nitro-L-arginine methyl ester (100 mol/L; Figure
5B) or exendin 9-39 (100 nmol/L) and GLP-1R antibody (2.5
g/mL; Figure 5C). By contrast, exendin 4 treatment for 12
hours had no effect on EDR in WKYs (Figure 5D).
Exendin 4 Increases AMPK and eNOS
Phosphorylations and Stimulates NO Production
in SHR Aortic Endothelial Cells
Exendin 4 (10 nmol/L) stimulated NO production in primary SHR
aortic endothelial cells, which was inhibited by pretreatment with
exendin 9-39 (100 nmol/L), SQ22536 (100 mol/L), H89 (1
mol/L), compound C (10 mol/L), or N
G
-nitro-L-arginine methyl
ester (100 mol/L; Figure S4 and S5). Twelve-hour ex vivo
treatment with either exendin 4 (10 nmol/L) or sitagliptin (10
mol/L) increased the cGMP level in SHR renal arteries (Figure
S6). 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 in
endothelial cells, whereas suppression of the AMPK activity by
dominant-negative AMPK2 (K45R mutated) inhibited such ef-
fects. The level of AMPK2 increased significantly in SHR
endothelial cells by expression of wild-type AMPK but not
dominant-negative AMPK (Figure S8).
Sitagliptin Ameliorates Endothelial Dysfunction in
Renal Arteries From Hypertensive Patients
EDRs were impaired in renal arteries from hypertensive
patients compared with those from normotensive patients,
whereas exendin 4 (10 nmol/L; 12 hours) improved EDRs
in 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 (Figure
6C through 6F) were elevated by exendin 4 in these arteries.
Discussion
The present study demonstrated a functional importance of
GLP-1 and GLP-1R in the regulation of endothelial func-
tion in SHR renal vasculature. The major novel findings
0
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)
*
#
Veh Sitagliptin
p-PKA C(Thr
197
)
PKA C
GAPDH
SHR
p-LKB1(Ser
334
)
LKB1
GAPDH
Veh Sitagliptin
SHR
- - - +
- - + -
CC
Ex9-39
- - - +
- - + -
CC
Ex9-39
0
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(
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)
#
Veh Sitagliptin
p-AMPK(Thr
172
)
AMPK
GAPDH
SHR
0
1
2
p
-
e
N
O
S
/
e
N
O
S
(
C
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p
a
r
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d

t
o

v
e
h
i
c
l
e
)
p-eNOS(Ser
1177
)
eNOS
GAPDH
Veh Sitagliptin
SHR
- - - +
- - + -
CC
Ex9-39
CC
Ex9-39
*
- - - +
- - + -
*
#
*
*
#
#
*
#
-9 -8 -7 -6 -5
0
50
100
Control
Compound C
Exendin 9-39
L-NAME
ACh (log mol/L)
R
e
l
a
x
a
t
i
o
n
(
%

P
h
e

t
o
n
e
)
SHR+sitagliptin
#
#
#
C A B
E D
Figure 4. Endothelial function improved by sitagliptin is glucagon-like peptide 1 receptor (GLP-1R) dependent in spontaneously
hypertensive 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 N
G
-nitro-L-arginine methyl
ester (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.
Liu et al DPP-4 Inhibition Restores Endothelial Function 837
at Chinese University of Hong Kong on August 30, 2012 http://hyper.ahajournals.org/ Downloaded from
include the following: (1) treatment with sitagliptin in vivo
and exendin 4 ex vivo improved endothelial function in
SHR renal arteries via the sequential activation of the
PKA/LKB1/AMPK/eNOS axis; (2) exendin 4 stimulated
NO production in SHR aortic endothelial cells and im-
proved endothelial function in renal arteries from hyper-
tensive patients; (3) GLP-1R expression was reduced in
SHR renal arteries, which was upregulated by chronic
sitagliptin treatment; and (4) 2-week oral administration of
sitagliptin to SHR lowered SBP and improved RBF with-
out affecting glucose metabolism (Figure S9). Our study
reveals a protective role of GLP-1 and related agents in
improving endothelial function in hypertension through
the activation of AMPK.
Clinical and experimental studies suggest that GLP-1 and
its analogs confer cardiovascular protection through favor-
able modulation of heart rate, blood pressure, and cardiac
hemodynamic responses.
15,23,24
The present study shows that
in vivo sitagliptin or in vitro exendin 4 treatment improved
the impaired EDR in SHR or hypertensive patients and
stimulated NO production in primary SHR aortic endothelial
cells, which is likely mediated by AMPK/eNOS activation.
To further confirm the role of AMPK, the present study
show that the suppression of AMPK2 activity by dominant-
negative AMPK2 in SHR endothelial cells inhibited the
AMPK/eNOS phosphorylations and NO production trig-
gered by exendin 4, whereas increasing the AMPK activity
by wild-type AMPK overexpression enhanced the stimula-
tory effect of exendin 4. AMPK, which is one of the principal
kinases 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 used
3 assay methods, including N
G
-nitro-L-arginine methyl ester
sensitive EDR in renal arteries, eNOS phosphorylation, and NO
production, to support that NO bioavailability was increased by
sitagliptin or exendin 4 treatment. NO levels in SHR endothelial
cells were detected by the use of 4-amino-5-methylamino-2=,7=-
difluorofluorescein fluorescence
29
and the Total Nitric Oxide
Assay kit.
30
After release, NO acts on vascular smooth muscle
cells to stimulate the activity of soluble guanylate cyclase, an
enzyme that catalyzes the chemical conversion of GTP into
cGMP.
31
Therefore, we measured cGMP levels in SHR renal
arteries that were elevated by 12-hour ex vivo incubation with
exendin 4 or sitagliptin and thus confirmed the increased NO
production (Figure S6). The present results suggest that GLP-
1elevating agents could be a novel upstream regulator of
AMPK to preserve the NO bioavailability and endothelial
function in hypertension.
GLP-1R is expressed in human coronary artery endothe-
lial cells,
32
as well as in the endothelium and smooth
muscle cells,
23
which were shown to be downregulated by
hyperglycemia, and this decrease likely contributed to the
impaired incretin effects in diabetes mellitus.
33
The pres-
ent study shows that GLP-1R expression was reduced in
SHR renal arteries, whereas in vivo sitagliptin treatment
increased the expression of GLP-1R, supporting that
DPP-4 inhibition restores the expression and function of
GLP-1/GLP-1R in SHR arteries. However, the mechanism
of GLP-1R downregulation in hypertension needs to be
further elucidated.
The actions of GLP-1R are thought to involve cAMP
production and PKA activation.
23,34
Kieffer and Habener
35
-9 -8 -7 -6 -5
0
50
100
Control
Exendin-4
WKY
ACh (log mol/L)
R
e
l
a
x
a
t
i
o
n
(
%

P
h
e

t
o
n
e
)
-9 -8 -7 -6 -5
0
50
100
Exendin-4
Control
H89+exendin-4
SQ22536+exendin-4
ACh (log mol/L)
R
e
l
a
x
a
t
i
o
n
(
%

P
h
e

t
o
n
e
)
SHR
*
#
#
A
-9 -8 -7 -6 -5
0
50
100
Exendin-4
Control
CC+exendin-4
L-NAME+exendin-4
SHR
ACh (log mol/L)
R
e
l
a
x
a
t
i
o
n
(
%

P
h
e

t
o
n
e
)
*
#
#
-9 -8 -7 -6 -5
0
50
100
Exendin-4
GLP-1R Ab+exendin-4
Control
Ex9-39+exendin-4
SHR
ACh (log mol/L)
R
e
l
a
x
a
t
i
o
n
(
%

P
h
e

t
o
n
e
)
*
#
#
B
D C
Figure 5. Exendin 4 ameliorates endothelial dysfunction in spontaneously hypertensive rat (SHR) renal arteries. Reversal of the
improved 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 N
G
-nitro-L-arginine methyl ester (l-NAME; 100
mol/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 EDR
in renal arteries from Wistar-Kyoto rats (WKYs). Data are meanSEM. *P0.05 vs control. #P0.05 vs exendin 4. n4 for WKYs; n6
for SHRs.
838 Hypertension September 2012
at Chinese University of Hong Kong on August 30, 2012 http://hyper.ahajournals.org/ Downloaded from
suggested the role of the GLP-1R and cAMP in the actions of
GLP-1 on vascular endothelium. Moreover, PKA stimulates
LKB1 for AMPK activation in hepatocytes.
36
The present
study demonstrated that sitagliptin stimulated the activation
of LKB1/AMPK subsequent to cAMP/PKA signaling on
activation of GLP-1R (Figure S10). Finally, the in vivo effect
of sitagliptin was assessed in SHRs by measuring RBF and
blood pressure. Chronic GLP-1 treatment lowers blood pres-
sure in patients with type 2 diabetes mellitus,
37
and exendin
4 also exerts an antihypertensive effect in salt-sensitive
hypertensive mice.
13,17
Another study suggests that DPP-4
inhibition by sitagliptin attenuates blood pressure elevation in
young SHRs by diminishing proximal tubule sodium reab-
sorption.
38
In the present study, we observe that 2-week
sitagliptin treatment reduced SBP in adult SHRs. The resto-
ration of AMPK/eNOS in SHR arteries after sitagliptin
treatment might also contribute in part to blood pressure
reduction.
39,40
Renal function is of importance in controlling
blood pressure in the development of essential hypertension,
3
and RBF is a parameter of renal function.
40
RBF is reduced
in renovascular beds in essential hypertension, which is
attributed to arteriolar constriction.
41
SHRs are known to
have higher mean arterial pressure and renal vascular resis-
tance than WKYs under baseline conditions. Moreover, NO
synthase inhibition induced hypertension
42
and caused more
reduction of RBF in SHRs,
43
suggesting that RBF is closely
related to the NO availability in renal circulation. The present
study showed that RBF was reduced in SHRs compared with
WKYs, and 2-week sitagliptin treatment increased RBF in
SHRs, which may in part contribute to the blood pressure
lowering effect of sitagliptin.
Perspectives
We demonstrate that DPP-4 inhibition by sitagliptin pre-
serves vascular GLP-1/GLP-1R function in SHRs, a genetic
model of hypertension. GLP-1induced AMPK/eNOS acti-
vation restores endothelium-dependent relaxation and RBF,
thus helping to reduce SBP in SHRs. Like sitagliptin, the
GLP-1R agonist exendin 4 is also effective in augmenting
endothelial function in hypertensive rats and patients, thereby
elucidating the mechanism underlying the vascular benefits
of GLP-1 and related agents. Taken together, the novel
findings of the present study highlight the prospect for the use
of GLP-1elevating agents and GLP-1R agonists against
vascular dysfunction in hypertension.
Acknowledgment
We thank Merck Research Laboratories for the generous gift of
sitagliptin for the present study.
Sources of Funding
This study is supported by the National Basic Research Program of
China (2012CB517805), research grants (466110 and HKU4/
CRF10) from the Research Grants Council of Hong Kong, and
Chinese University of Hong Kong Focused Investment Scheme B.
0.0
0.5
1.0
p
L
K
B
1
/
L
K
B
1
(
C
o
m
p
a
r
e
d

t
o

N
T
)
0.0
0.5
1.0
p
-
e
N
O
S
/
e
N
O
S
(
C
o
m
p
a
r
e
d

t
o

N
T
)
0.0
0.5
1.0
p
-
A
M
P
K
/
A
M
P
K

(
C
o
m
p
a
r
e
d

t
o

N
T
)
0.0
0.5
1.0
G
L
P
-
1
R
/
G
A
P
D
H
(
C
o
m
p
a
r
e
d

t
o

N
T
)
-9 -8 -7 -6 -5
0
50
100
HT
HT+exendin-4
Renal arteries from patients
NT
ACh (log mol/L)
R
e
l
a
x
a
t
i
o
n
(
%

P
h
e

t
o
n
e
)
*
#
p-AMPK (Thr
172
)
AMPK
GAPDH
p-eNOS (Ser
1177
)
eNOS
GAPDH
GLP-1R
GAPDH
*
#
*
#
*
#
D
E
B C A
NT C E
HT
p-PKA C(Thr
197
)
PKA C
GAPDH
p-LKB1(Ser
334
)
LKB1
GAPDH
0.0
0.5
1.0
p
P
K
A

C
/
P
K
A

C

(
C
o
m
p
a
r
e
d

t
o

N
T
)
*
#
*
#
F
NT C E
HT
NT C E
HT
NT C E
HT
NT C E
HT
Figure 6. Exendin 4 ameliorates endothelial dysfunction in renal arteries from hypertensive patients. A, Endothelium-dependent
relaxation (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), and
endothelial 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; n6
for NT relaxations.
Liu et al DPP-4 Inhibition Restores Endothelial Function 839
at Chinese University of Hong Kong on August 30, 2012 http://hyper.ahajournals.org/ Downloaded from
Disclosures
None.
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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 DPP-4 Inhibition Restores Endothelial Function 841
at Chinese University of Hong Kong on August 30, 2012 http://hyper.ahajournals.org/ Downloaded from
Liu et al., 2012, Supplemental Materials
1

Dipeptidyl Peptidase-4 Inhibitor Sitagliptin Protects Endothelial Function
in Hypertension through GLP-1 Dependent Mechanism

Limei Liu
1,2
, Jian Liu
1
, Wing Tak Wong
1
, Xiao Yu Tian
1
, Chi Wai Lau
1
, Yi-Xiang Wang
3
, Gang
Xu
4
, Yunfei Pu
5
, Zhiming Zhu
5
, Aimin Xu
6,7
, Karen SL Lam
6
, Zhen Yu Chen
8
, Chi Fai Ng
9
,
Xiaoqiang Yao
1
, Yu Huang
1

1
Institute of Vascular Medicine, Li Ka Shing Institute of Health Sciences, and School of Biomedical
Sciences, Chinese University of Hong Kong, Hong Kong, China;
2
Department of Physiology and
Pathophysiology, Peking University Health Science Center, Peking, China;
3
Department of Imaging
and Interventional Radiology , Chinese University of Hong Kong, Hong Kong, China;
4
Department of
Medicine and Therapeutics, Chinese University of Hong Kong, Hong Kong, China;
5
Department of
Hypertension and Endocrinology, Daping Hospital, Third Military Medical University, China;
Departments of
6
Medicine and
7
Pharmacology and Pharmacy, University of Hong Kong, Hong Kong,
China;
8
School of Life Sciences and
9
Department of Surgery, Chinese University of Hong Kong, Hong
Kong, China
Correspondence: Yu Huang (yu-huang@cuhk.edu.hk) or Limei Liu (liu_limei@126.com)


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)
at Chinese University of Hong Kong on August 30, 2012 http://hyper.ahajournals.org/ Downloaded from
Liu et al., 2012, Supplemental Materials
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 CO
2
suffocation and intrarenal
arteries were removed and placed in ice-cold Krebs
solution (mmol/L): 119 NaCl, 4.7 KCl, 2.5 CaCl
2
,

1
MgCl
2
, 25 NaHCO
3
, 1.2 KH
2
PO
4
, 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% O
2
-5% CO
2
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
at Chinese University of Hong Kong on August 30, 2012 http://hyper.ahajournals.org/ Downloaded from
Liu et al., 2012, Supplemental Materials
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, Thr
197
), phospho-LKB1 (Ser
334
), phospho-
eNOS (Ser
1177
), phospho-AMPK (Thr
172
), 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 (F
1
/F
0
), 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 (Ser
1177
), anti-eNOS, anti-GLP-1
receptor and anti-GLP-1 antibodies were obtained
from Abcam (Cambridge, MA). Anti-phospho-PKA C
(Thr
197
), phosphor-LKB1 (Ser
334
), phospho-AMPK
(Thr
172
), 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 (pD
2
) and
at Chinese University of Hong Kong on August 30, 2012 http://hyper.ahajournals.org/ Downloaded from
Liu et al., 2012, Supplemental Materials
4
the maximum relaxation (E
max
%) 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<0.05 indicates
statistically significant difference.

Additional Results
Telemetry results showed that systolic blood pressure
(SBP) gradually reduced about one week after
sitagliptin treatment in SHRs comparing to that before
treatment or that of SHR treated with vehicle at the
same time. Mean arterial pressure (MBP) also
reduced in SHRs after sitagliptin treatment comparing
to that before treatment. Diastolic BPs were similar in
all the groups. No obvious diurnal rhythm of BP or HR
was found in all the groups of rats (Supplemental Fig.
S1C).

Additional Discussion
However, the effect of DPP-4 inhibitors on arterial
blood pressure is still disputed. DPP-4 inhibition has
no and can either lower or elevate blood pressure
depending on types of inhibitors, the duration of
treatment, and the level of background anti-
hypertensive treatment.
7, 8
This variation in the impact
on blood pressure is likely related to the fact that
multiple substrates are catalyzed by DPP-4. Thus, the
advantages and disadvantages of the use of DPP-4
inhibitors with respect to the cardiovascular function
can be influenced by the relative amount of various
bioactive substrates such as vasoconstrictive
neuropeptide Y, their action on sympathetic nerves,
and hemodynamic interaction with other blood
pressure regulators such as angiotensin-converting
enzyme.
9
In this study, we only observed reduction of
systolic blood pressure during 14-day treatment with
sitagliptin as measured by telemetry, without
significant change of diastolic and mean arterial
pressure. Further detailed study on blood pressure
regulation by DPP-4 inhibitor may be useful to provide
more information on blood pressure control in different
animals under normotensive or hypertensive
conditions.

Limitations of the present study
The present study demonstrates that DPP-4 inhibition
improves endothelium-dependent relaxations in renal
interlobar arteries of SHR. The effect of DPP-4
inhibition is not examined in resistance arteries which
also contribute to the regulation of blood pressure. In
addition, whether DPP-4 inhibition also decreases
vascular inflammation or whether it improves renal
function through other mechanisms that are
associated with blood pressure regulation are not
studied. There are other methods which can measure
the vasomotor response of renal arterioles,
10, 11

which are more important for blood pressure
regulation, so this method will be employed to
observe the response of arterioles to GLP-1 in the
future investigations. However, this is beyond the
scope of the present study.

References
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Kroetsch JT, Rush JW. AMP-activated protein
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2. Leung FP, Yao X, Lau CW, Ko WH, Lu L, Huang
Y. Raloxifene relaxes rat intrarenal arteries by
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3. Wong WT, Tian XY, Chen Y, Leung FP, Liu L,
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4. Jin J, Mullen TD, Hou Q, Bielawski J, Bielawska
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FP, Flatt PR. Chronic treatment with exendin(9-
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abnormalities of obesity-related diabetes in ob/ob
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6. Han WQ, Wong WT, Tian XY, Huang Y, Wu LY,
Zhu DL, Gao PJ. Contributory role of
endothelium and voltage-gated potassium
channels in apocynin-induced vasorelaxations. J
Hypertens. 2010;28:2102-2110.
7. Pacheco BP, Crajoinas RO, Couto GK, Davel AP,
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peptidase IV inhibition attenuates blood pressure
rising in young spontaneously hypertensive rats.
J Hypertens. 2011;29:520-528.
8. Jackson EK, Dubinion JH, Mi Z. Effects of
dipeptidyl peptidase IV inhibition on arterial blood
pressure. Clin Exp Pharmacol Physiol.
2008;35:29-34.
9. Marney A, Kunchakarra S, Byrne L, Brown NJ.
Interactive hemodynamic effects of dipeptidyl
peptidase-IV inhibition and angiotensin-
converting enzyme inhibition in humans.
Hypertension. 2010;56:728-733.
10. Al-Mashhadi RH, Skott O, Vanhoutte PM,
Hansen PB. Activation of A(2) adenosine
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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.

at Chinese University of Hong Kong on August 30, 2012 http://hyper.ahajournals.org/ Downloaded from
Liu et al., 2012, Supplemental Materials
6
Supplemental Figures
A B


C

0 6 12 18 24
100
150
200
250
Day 0
SHR Sitagliptin
SHR Vehicle
WKY Vehicle
WKY Sitagliptin
Time (hour)
S
B
P

(
m
m
H
g
)
0 6 12 18 24
100
150
200
250
Day 14
SHR Sitagliptin
SHR Vehicle
WKY Vehicle
WKY Sitagliptin
Time (hour)
S
B
P

(
m
m
H
g
)
0 7 14
100
140
180
220
WKY Vehicle
WKY Sitagliptin
SHR Vehicle
SHR Sitagliptin
Time (Day)
S
B
P

(
m
m
H
g
)
0 6 12 18 24
75
100
125
150
175
Day 0
SHR Sitagliptin
SHR Vehicle
WKY Vehicle
WKY Sitagliptin
Time (hour)
D
B
P

(
m
m
H
g
)
0 6 12 18 24
75
100
125
150
175
Day 14
SHR Sitagliptin
SHR Vehicle
WKY Vehicle
WKY Sitagliptin
Time (hour)
D
B
P

(
m
m
H
g
)
0 7 14
80
100
120
140
WKY Vehicle
WKY Sitagliptin
SHR Vehicle
SHR Sitagliptin
Time (day)
D
B
P

(
m
m
H
g
)
0 6 12 18 24
75
100
125
150
175
Day 0
SHR Sitagliptin
WKY Sitagliptin
SHR Vehicle
WKY Vehicle
Time (hour)
M
A
B
P

(
m
m
H
g
)
0 6 12 18 24
75
100
125
150
175
Day 14
SHR Sitagliptin
WKY Sitagliptin
SHR Vehicle
WKY Vehicle
Time (hour)
M
A
B
P

(
m
m
H
g
)
0 7 14
75
100
125
150
175
WKY Vehicle
WKY Sitagliptin
SHR Vehicle
SHR Sitagliptin
Time (day)
M
A
B
P

(
m
m
H
g
)
0 6 12 18 24
200
300
400
500
Day 0 WKY Vehicle
SHR Vehicle
SHR Sitagliptin
WKY Sitagliptin
Time (hour)
H
e
a
r
t

r
a
t
e

/
m
i
n
0 6 12 18 24
200
300
400
500
Day 14 WKY Vehicle
WKY Sitagliptin
SHR Vehicle
SHR Sitagliptin
Time (hour)
H
e
a
r
t

r
a
t
e

/
m
i
n
0 7 14
200
300
400
500
WKY Vehicle
SHR Vehicle
SHR Sitagliptin
WKY Sitagliptin
Time (day)
H
e
a
r
t

r
a
t
e

/
m
i
n
SBP SBP DBP MABP HR

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<0.05 vs WKY vehicle, #p<0.05 vs SHR vehicle. n=4.
at Chinese University of Hong Kong on August 30, 2012 http://hyper.ahajournals.org/ Downloaded from
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7

























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<0.05 vs WKY
vehicle, #p<0.05 vs SHR vehicle. n=6 for Western blotting; n=8 for WKY ELISA;
n=10 for SHR ELISA.













Figure S3. Levels of cAMP in SHR renal arteries. Chronic sitagliptin
treatment increased cAMP levels in SHR renal arteries, which were inhibited
by 30 min-incubation with exendin 9-39 (100 nmol/L) and SQ22536 (100
mol/L) but not by compound C (10 mol/L). Data are meansSEM. *p<0.05
vs vehicle, #p<0.05 vs sitagliptin. n=6.


0
10
20
30
40
a b c d e
a: Vehicle
b: Sitagliptin
c: Exendin 9-39 + sitagliptin
d: SQ22536 +sitagliptin
e: Compound C + sitagliptin
c
A
M
P

l
e
v
e
l
(
p
m
o
l
/
m
g

p
r
o
t
e
i
n
)
#
*
#
0
10
20
30
40
a b c d e
a: Vehicle
b: Sitagliptin
c: Exendin 9-39 + sitagliptin
d: SQ22536 +sitagliptin
e: Compound C + sitagliptin
c
A
M
P

l
e
v
e
l
(
p
m
o
l
/
m
g

p
r
o
t
e
i
n
)
#
*
#
A
B
0
1
2
G
L
P
-
1
/
G
A
P
D
H
(
C
o
m
p
a
r
e
d

t
o

W
K
Y

v
e
h
i
c
l
e
)
GLP-1
GAPDH
#
*
- + - +
WKY SHR
- + - +
WKY SHR
Sitagliptin
0
10
20
30
40
50
G
L
P
-
1

c
o
n
c
e
n
t
r
a
t
i
o
n
i
n

p
l
a
s
m
a

(
p
m
o
l
/
L
)
WKY SHR
#
*
- + - + Sitagliptin
0
10
20
30
40
50
G
L
P
-
1

c
o
n
c
e
n
t
r
a
t
i
o
n
i
n

p
l
a
s
m
a

(
p
m
o
l
/
L
)
WKY SHR
#
*
- + - + Sitagliptin
0.0
0.5
1.0
1.5
G
L
P
-
1
R
/
G
A
P
D
H
(
C
o
m
p
a
r
e
d

t
o

W
K
Y

v
e
h
i
c
l
e
)
GLP-1R
GAPDH
#
*
Sitagliptin - + - +
WKY SHR
Sitagliptin - + - +
WKY SHR
C
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8




































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<0.05 vs control. #p<0.05
vs exendin-4. n=6 for control; n=4 for various treatments.





0 10 20
1
2
3
Exendin-4
Control
WT+exendin-4
DN+exendin-4
Time (min)
F
1
/
F
0
(
D
A
F
-
D
A

f
l
u
o
r
e
s
c
e
n
c
e
)
0 10 20
1
2
3
Exendin-4
Control
CC+exendin-4
L-NAME+exendin-4
Time (min)
F
1
/
F
0
(
D
A
F
-
D
A

f
l
u
o
r
e
s
c
e
n
c
e
)
0 10 20
1
2
3
Exendin-4
Control
H89+exendin-4
SQ22536+exendin-4
Time (min)
F
1
/
F
0
(
D
A
F
-
D
A

f
l
u
o
r
e
s
c
e
n
c
e
)
0 10 20
1
2
3
Exendin-4
Ex9-39+exendin-4
Control
Time (min)
F
1
/
F
0
(
D
A
F
-
D
A

f
l
u
o
r
e
s
c
e
n
c
e
)
Exendin-4 (10 nmol/L)
Exendin-4
Ex9-39+ex4
CC +ex4
L-NAME+ex4
WT+ex4
DN+ex4
Time-matched
control
High NO
Low NO
High NO
Low NO
0 min 20 min
H89+ex4
SQ22536+ex4
#
#
*
#
*
#
*
#
#
*
#
Bar:100 m
*
A
B
C
D
E
at Chinese University of Hong Kong on August 30, 2012 http://hyper.ahajournals.org/ Downloaded from
Liu et al., 2012, Supplemental Materials
9


















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<0.05 vs control. #p<0.05 vs exendin-4. n=5.



















Figure S6. cGMP levels in SHR renal arteries. Incubation with sitagliptin (10
mol/L, 12 hours) or exendin-4 (10 nmol/L, 12 hours) increased cGMP levels in
SHR renal arteries. Data are meansSEM. *p<0.05 vs vehicle. n=4.



0
60
120
1 2 3 4 5 6
1, Control
2, Exendin-4
3, Exendin 9-39+exendin-4
4, SQ22536+exendin-4
5, H89+exendin-4
6, Compound C+exendin-4
N
i
t
r
a
t
e

a
n
d

n
i
t
r
i
t
e

c
o
n
c
.
(
n
m
o
l
/
m
g

p
r
o
t
e
i
n
)
*
#
#
#
#
0.0
3.5
7.0
1 2 3
1, Control
2, Sitagliptin
3, Exendin-4
c
G
M
P

l
e
v
e
l
(
p
m
o
l
/
m
g

p
r
o
t
e
i
n
)
*
*
at Chinese University of Hong Kong on August 30, 2012 http://hyper.ahajournals.org/ Downloaded from
Liu et al., 2012, Supplemental Materials
10




















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<0.05 vs
control (C). #p<0.05 vs exendin-4. n=6.



















Figure S8. Levels of AMPK2 in cultured SHR aortic endothelial cells .
The level of AMPK2 increased by expression of WT-AMPK (WT) and
unaffected by expression of DN-AMPK (DN) compared with control (C, Cont).
Data are meansSEM. *p<0.05 vs control. #p<0.05 vs WT. n=4.

Cont WT DN
0
1
2
3
A
M
P
K

2
/
G
A
P
D
H
(
C
o
m
p
a
r
e
d

t
o

c
o
n
t
r
o
l
)
C C WT WT DN DN
AMPK2
GAPDH
*
#
0
1
2
p
-
A
M
P
K
/
A
M
P
K

(
C
o
m
p
a
r
e
d

t
o

c
o
n
t
r
o
l
)
*
#
*
C C WT WT DN DN
Exendin-4 - + - + - +
p-AMPK (Thr
172
)
AMPK
GAPDH
#
p-eNOS (Ser
1177
)
eNOS
GAPDH
C C WT WT DN DN
Exendin-4 - + - + - +
0
1
2
p
-
e
N
O
S
/
e
N
O
S
(
C
o
m
p
a
r
e
d

t
o

c
o
n
t
r
o
l
)
*
#
#
*
A B
at Chinese University of Hong Kong on August 30, 2012 http://hyper.ahajournals.org/ Downloaded from
Liu et al., 2012, Supplemental Materials
11















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.


LKB1
Sitagliptin
GLP-1 receptor
ATP
cAMP
AMPK
eNOS
L-citrulline
L-arginine
NO
Endothelial cells Relaxation
S
m
o
o
t
h

m
u
s
c
l
e

c
e
l
l
s
PKA
G
GTP
AC

GLP-1
ITT (1 unit/kg)
0 30 60 90 120
20
70
120
WKY vehicle
SHR vehicle
SHR+sitagliptin
Time (min)
P
l
a
s
m
a

g
l
u
c
o
s
e
(
%

o
f

i
n
i
t
i
a
l
)
at Chinese University of Hong Kong on August 30, 2012 http://hyper.ahajournals.org/ Downloaded from