Metformin reduces blood pressure and restores endothelial function in aorta

of streptozotocin-induced diabetic rats

Jayesh B. Majithiya
*
, R. Balaraman
Pharmacy Department, Faculty of Technology and Engineering, M. S. University of Baroda, Kalabhavan, Baroda-390001, Gujarat, India
Received 21 July 2005; accepted 11 October 2005
Abstract
Effect of metformin treatment on blood pressure, endothelial function and oxidative stress in streptozotocin (STZ)-induced diabetes in rats was
studied. In vitro effect of metformin on vascular reactivity to various agonist in the presence of metformin in untreated nondiabetic and STZ-
diabetic rats were also studied. Sprague-Dawley rats were randomized into nondiabetic and STZ-diabetic groups. Rats were further randomized to
receive metformin (150 mg/kg) or vehicle for 4 weeks.
Metformin treatment reduced blood pressure without having any significant effect on blood glucose level in STZ-diabetic rats. Enhanced
phenylephrine (PE)-induced contraction and impaired acetylcholine (Ach)-induced relaxation in STZ-diabetic rats were restored to normal by
metformin treatment. Enhanced Ach-induced relaxation in metformin-treated STZ-diabetic rats was blocked due to pretreatment with 100 AM of
Nx-nitro-l-arginine-methyl ester (l-NAME) or 10 AM of methylene blue but not 10 AM of indomethacin. Metformin treatment significantly
increased antioxidant enzymes and reduced lipid peroxidation in STZ-diabetic rats. In vitro studies in aortic rings of untreated nondiabetic and
STZ-diabetic rats showed that the presence of higher concentration of metformin (1 mM and 10 mM) significantly reduced PE-induced
contraction and increased Ach-induced relaxation. Metformin per se relaxed precontracted aortic rings of untreated nondiabetic and STZ-diabetic
rats in a dose-dependent manner. Pretreatment with l-NAME or removal of endothelium blocked metformin-induced relaxation at lower
concentration (up to 30 AM) but not at higher concentration (above 30 AM). Metformin-induced relaxation was blocked in the presence of 1 mM
of 4-aminopyridine, or 1 mM of tetraethylammonium but not in the presence of 100 AM of barium ion or 10 AM of glybenclamide. The restored
endothelial function along with direct effect of metformin on aortic rings and reduced oxidative stress contributes to reduced blood pressure in
STZ-diabetic rats. From the present study, it can be concluded that metformin administration to STZ-diabetic rats lowers blood pressure, and
restores endothelial function.
D 2005 Elsevier Inc. All rights reserved.
Keywords: Metformin; Streptozotocin-induced diabetes; Endothelial function; Blood pressure; Oxidative stress
Introduction
Cardiovascular disease is one of the leading causes of
death in the western world and diabetes mellitus, which alters
the vascular responsiveness to several vasoconstrictors and
vasodilators, is a major factor underlying its development
(Senses et al., 2001). Most of the complications in diabetes
are due to increased serum glucose and increased generation
of oxygen-derived free radicals, which lead to endothelium
dysfunction. It has been shown that vessels from diabetic
animals, exhibited abnormal endothelium-dependent vascular
relaxation to acetylcholine (Oyama et al., 1986; Kamata et al.,
1989).
Metformin belongs to bigunide insulin-sensitizing class of
anti-diabetic drugs, widely used for the treatment of type 2
diabetes. The exact mechanisms of action of metformin, is
poorly understood (Moller, 2001), but it includes suppression
of endogenous glucose output by liver and increased
sensitivity in skeletal muscle (Matthaei et al., 2000).
Metformin lowers blood pressure in certain human patients
(Landin et al., 1991; Giugliano et al., 1993) but not in others
(Calle-Pascual et al., 1995; GudbjoRnsdottir et al., 1994;
Campbell et al., 1987). Metformin lowers blood pressure in
0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.lfs.2005.10.020
* Corresponding author. Tel.: +91 265 2434187; fax: +91 265 2418927.
E-mail addresses: jayeshbm@yahoo.com (J.B. Majithiya),
rbalaraman2000@yahoo.com (R. Balaraman).
Life Sciences 78 (2006) 2615 2624
www.elsevier.com/locate/lifescie
fructose fed rats (Verma et al., 1994), OLETF rats
(Kosegawa et al., 1996), SHR (Bhalla et al., 1996; Muntzel
et al., 1999) and insulin-resistant rats (Katakam et al., 2000).
Metformin also has antioxidant activity which is independent
of its effect of insulin activity (Faure et al., 1999). Recently
Sartoretto et al. (2005) has reported that metformin increases
nitric oxide activity but not expression and that it improves
microvascular reactivity in n- STZ-diabetic rats (Type 2).
There are no reports on effect of metformin on STZ-diabetic
model (Type I) wherein free radical generation is one of the
main causes of endothelial dysfunction (Kobayashi and
Kamata, 1999; Zanetti et al., 2001). We have previously
shown that administration of STZ causes a significant
increase in blood pressure and oxidative stress (Majithiya
et al., 2005). The current study investigates the effect of
chronic metformin treatment on blood pressure, endothelial
function and oxidative stress in streptozotocin-induced
diabetic rats. Moreover, in vitro effects of metformin on
aortic rings of STZ-diabetic and nondiabetic rats are also
studied.
Materials and methods
Drugs
Metformin hydrochloride and glybenclamide were
obtained as a gift sample from Alembic Ltd, Baroda.
Streptozotocin, phenylephrine, acetylcholine, Nx-nitro-l-ar-
ginine-methyl ester (l-NAME), indomethacin, 4-aminopyr-
idine, tetraethylammonium (TEA), epinephrine, 1,1,3,3,-tetra
ethoxy propane, superoxide dismutase, catalase and glutathi-
one standard were obtained form SIGMA, St. Louis, MO,
USA. All other chemicals and reagents used in the study
were of analytical grade. The composition of the Krebs
solution (mM) was NaCl 118, KCl 4.7, CaCl
2
2.5, MgSO
4
1.2, KH
2
PO
4
1.2, NaHCO
3
22.0, and glucose 11.0. Stock
solutions of drugs were made by dissolving drugs in double
distilled water, except glybenclamide and indomethacin,
which were dissolved in DMSO and Na
2
CO
3
solution,
respectively.
Experimental protocol
All experiments and protocols described in present study
were approved by the Institutional Animal Ethics Committee
(IAEC) of M.S. University, Baroda and are in accordance
with guidelines as per Guide for the care and use of
laboratory animals published by NIH publication (No. 85-23
revised 1996) and with permission from Committee for the
Purpose of Control and Supervision of Experiments on
Animals (CPCSEA), Ministry of Social Justice and Empow-
erment, Government of India. Male Sprague-Dawley rats
(200T15 g) were housed as 3 animals per group and
maintained under standardized condition (12-h light/dark
cycle, 24 -C) and provided free access to pelleted CHAK-
KAN diet (Nav Maharashtra Oil Mills Pvt. Ltd., Pune) and
purified drinking water ad libitum. Diabetes was induced by
single intravenous injection of streptozotocin (55 mg/kg,
STZ) dissolved in normal saline. The control animals were
injected with equal volume of vehicle. After 3 days following
streptozotocin administration, blood was collected from tail
vein and serum samples were analyzed for blood glucose
(Enzymatic kits, GOD/POD method, SPAN diagnostics Pvt.
Ltd, India). Animals showing fasting blood glucose higher
than 250 mg/dl were considered as diabetic rats and used for
the study. Four weeks after induction of diabetes, blood
pressure was measured through tail cuff method, and rats with
systolic blood pressure higher than 135 mmHg were selected,
randomized into diabetic groups. On the other hand, age-
matched nondiabetic rats were randomized in to nondiabetic
groups. Nondiabetic control (n =16, N), STZ-diabetic control
(n =16, D), nondiabetic group treated with metformin (150
mg/kg/day, in drinking water) for 4 weeks (n =16, N-MET)
and STZ-diabetic rats treated with metformin (150 mg/kg/day,
in drinking water) for 4 weeks (n =16, D-MET). Blood
pressure was measured non-invasively at the start of study
and at weekly intervals by tail cuff method using LE 5002
storage pressure meter (LETICA scientific instruments, Spain)
in all the above-mentioned groups. For the blood pressure
measurements, animals were trained for at least 1 week until
blood pressure was steadily recorded with minimal stress and
restraint. A mean of 78 measurements of trained animals
was recorded.
Effect of PE and Ach on aortic rings obtained from control and
metformin-treated rats
After 4 weeks of treatment, thoracic aorta of all the
groups were isolated and mounted as previously described
(Majithiya et al., 2005). The aortic rings were connected to
isometric force displacement transducer connected to Gemini
pen recorder (UGO-BASILE, Italy), under tension of 2 g for
90 min before initiating experimental protocol. The presence
of functional endothelium was assessed by the ability of
acetylcholine (Ach, 0.1 AM) to induce more than 60% of
relaxation of rings precontracted submaximally with PE.
Aortic rings were considered denuded when there was less
than 10% relaxation to Ach. Concentrationresponse curves
to increasing concentrations of PE (1 nM10 AM) were
performed in rings with intact endothelium. Concentration
response curve of PE was also recorded in the presence and
absence of 100 AM of l-NAME. Indomethacin (10 AM) was
added to prevent the involvement of prostaglandins. Endo-
thelium-mediated relaxation was measured as a concentra-
tionresponse curve to Ach (1 nM10 AM) in rings
precontracted with PE (8090% of maximum response).
Endothelium-independent aortic relaxation to sodium nitro-
prusside (SNP=0.001 nM10 AM) was also measured in
rings with denuded endothelium. Concentration-dependent
relaxation to Ach was recorded in precontracted rings 30 min
after incubation with and in continued presence of 10 AM
indomethacin (a non-selective cyclo-oxygenase inhibitor), 10
AM of methylene blue (cGMP inhibitor) and 100 AM of l-
NAME (a non-selective nitric oxide synthase inhibitor) +10
J.B. Majithiya, R. Balaraman / Life Sciences 78 (2006) 26152624 2616
AM indomethacin. To investigate the effect of metformin
treatment on tone-related basal nitric oxide release, aortic
rings were submaximally (about 3035%) contracted with
PE (3 AM) and then response to the addition of l-NAME
(1100 AM) was recorded as described by Hayashi et al.
(1992).
Measurement of superoxide dismutase, catalase, reduced
glutathione and lipid peroxidation
After 4 weeks of treatment, animals were sacrificed. Liver,
kidney and aorta were isolated and weighed (Bafna and
Balaraman, 2004). The tissues were finely sliced and
homogenized in chilled Tris buffer at a concentration of
10% (w/v). The homogenates were centrifuged at 10,000g
at 0 -C for 20 min using Remi C-24 high speed cooling
centrifuge. The clear supernatant was used for estimation for
assays of lipid peroxidation (MDA content), endogenous
antioxidant enzymes (superoxide dismutase (SOD) and
catalase (CAT)) and reduced glutathione (GSH)). Superoxide
dismutase was determined by the method of Mishra and
Fridovich (1972). Catalase was estimated by the method
given by Aebi (1984). Reduced glutathione was determined
by the method of Moron et al. (1979). Lipid peroxidation or
malondialdehyde formation was estimated by the method of
Slater and Sawyer (1971).
Aortic nitrite levels
Nitrite was estimated colorimetrically with the Griess
reagent (Guevara et al., 1998) in aortic homogenate as
previously described (Majithiya et al., 2005). Briefly equal
volumes of aortic homogenate and Griess reagent (sulfanil-
amide 1% w/v, naphthylethylenediamine dihydrochloride 0.1%
w/v, and orthophosphoric acid 2.5% v/v) were mixed and
incubated at room temperature for 10 min and the absorbance
was determined at 540 nm wavelength and compared to those
of known concentrations of sodium nitrite. The amount of
nitrite formed was normalized to the protein content of the
respective aorta.
Effect of presence of metformin on PE, Ach and SNP dose
response curves on aortic rings of untreated non diabetic and
STZ-diabetic animals
Aortic rings of untreated age-matched nondiabetic
(n =28) and STZ-diabetic (n =28) were mounted in organ
bath as previously described (Majithiya et al., 2005).
Concentrationresponse curves to PE and Ach were
recorded in the presence or absence of metformin (0.1
AM100 mM). Relaxation to SNP (1 pM100 AM) in
endothelium denuded rings was also measured after metfor-
min incubation.
Effect of metformin per se on aortic rings of untreated animals
To find out the mechanism of metformin-induced relaxation,
concentration-dependent relaxation of metformin (10 nM100
mM) in PE contracted rings with intact and denuded
endothelium were recorded after 30 min incubation and in
continued presence of 100 AM of l-NAME, 10 AM of
indomethacin and 10 AM of methylene blue. Concentration-
dependent relaxation of metformin (10 nM10 mM) were also
Table 1
Blood glucose level and body weight of nondiabetic control (N), diabetic
control (D), nondiabetic animals treated with metformin (N-MET) and diabetic
animals treated with metformin (D-MET) rats before treatment (initial) and
after treatment (final) with metformin
Groups N D N-MET D-MET
Blood glucose level (mg/dl)
Initial 89T4.1 447T26.5
a
95T5.4
b
453T21.8
a
Final 91T8.2 458T19.2
a
91T12.3
b
421T24.2
a
Body weight (g)
Initial 211T12.8 215T11.7 217T9.6 212T8.7
Final 245T18.4 227T18.5 240T12.5 230T23.6
Systolic blood pressure (mmHg)
Initial 117T2.1 139T3.1
a
118T2.4
b
138T3.2
a
Final 120T2.4 143T4.5
a
119T2.5
b
122T2.6
b
Values are expressed as meanTS.E.M.
a
p <0.05, compared to nondiabetic (N) control group.
b
p <0.05, compared to diabetic control (D) group.
0.0
0.5
1.0
1.5
2.0
a
*
*
-9 -8 -7 -6 -5
0.0
0.5
1.0
1.5
2.0
D + L-NAME
D
D-MET + L-NAME
D-MET
b
*
#
@
log [PE](M)
T
e
n
s
i
o
n

(
g
)
T
e
n
s
i
o
n

(
g
)
N + L-NAME
N
N-MET + L-NAME
N-MET
Fig. 1. Concentration response curve of phenylephrine on aortic rings obtained
from (a) N ( r), N-MET ( 0) and (b) D ( ?), D-MET ( n) group in
presence (light legends) and absence of 100 AM of l-NAME (dark legends).
Values are expressed as meanTS.E.M. *p <0.05, compared to presence of l-
NAME;
#
p <0.05, compared to D;
@
p <0.05, compared to N (N=Normal,
D=Diabetic).
J.B. Majithiya, R. Balaraman / Life Sciences 78 (2006) 26152624 2617
recorded in PE contracted rings (with intact and denuded
endothelium) after 30 min incubation and in continued
presence of 1 mM of 4-aminopyridine (voltage-dependent K
+
channel inhibitor, K
v
), 1 mM of tetraethylammmonium (TEA,
non-specific calcium-activated K
+
channel inhibitor, K
Ca+
),
100 AM barium ion (Ba
2+
, inward rectifier K
+
channel
inhibitor, K
IR
) and 10 AM glybenclamide (ATP-sensitive K
+
channel inhibitor, K
ATP
).
Statistical analysis
All the data are expressed as meanTS.E.M. Data were
analyzed by ANOVA or two-way ANOVA for repeated
measurements followed by Bonferronis multiple comparison
tests as applicable. Differences were considered to be
statistically significant when p <0.05. The agonist pD
2
value
(log EC
50
) was calculated from concentrationresponse
curve by non-linear regression analysis of the curve using
computer based fitting program (Prism, GraphPad).
Results
Blood glucose, body weight and systolic blood pressure
All streptozotocin-injected animals developed diabetes.
The changes in blood glucose levels are shown in Table 1.
Blood glucose levels remained unchanged in nondiabetic
-9 -8 -7 -6 -5
0
20
40
60
80
100
120
N
D
N-MET
D-MET
*
*
*
log [Ach] (M)

%

R
e
l
a
x
a
t
i
o
n
Fig. 2. Concentration dependent relaxation of acetylcholine on aortic rings
obtained from N ( g), D ( ?), N-MET ( >) and D-MET ( n) groups
on aortic rings (with intact endothelium) precontracted with PE. Tension is
expressed as % relaxation on initial contraction with PE. Values are expressed as
meanTS.E.M. *p <0.05, compared to D (N=Normal, D=Diabetic).
0
20
40
60
80
100
*
*
a

%

R
e
l
a
x
a
t
i
o
n
*
*
b
-9 -8 -7 -6 -5
0
20
40
60
80
100
Ach + Indomethacin + L-NAME
Ach
Ach + Indomethacin
Ach + methylene blue
*
*
log [Ach] (M)

%

R
e
l
a
x
a
t
i
o
n
c
-9 -8 -7 -6 -5
*
*
d
log [Ach] (M)
Fig. 3. Concentration dependent relaxation of acetylcholine alone ( ?) and in presence of 10 AM of indomethacin ( g), 100 AM of l-NAME+10 AM of
indomethacin ( >) and 10 AM methylene blue ( g) in aortic rings (with intact endothelium) obtained from (a) N, (b) N-MET, (c) D and (d) D-MET group.
Tension is expressed as percentage relaxation of initial response to PE. Values are expressed as meanTS.E.M. *p <0.05, compared to Ach+methylene blue and
Ach+indomethacin+l-NAME (N=Normal, D=Diabetic).
J.B. Majithiya, R. Balaraman / Life Sciences 78 (2006) 26152624 2618
animals (N and N-MET groups). There was significant
( p <0.05) increase in blood glucose levels in streptozotocin
injected animals. Metformin treatment did not have any
significant effect on blood glucose level and body weight of
diabetic rats (Table 1). There was a significant ( p <0.05)
increase in systolic blood pressure in diabetic group as
compared to nondiabetic group (Table 1). Metformin (150
mg/kg/day) treatment for 4 weeks significantly reduced
systolic blood pressure of diabetic group (D-MET) as
compared to diabetic control (D) group. There was no
significant change in systolic blood pressure of nondiabetic
animals treated with metformin.
Contractile response to PE on aorta obtained from control
and metformin-treated rats in the presence and absence of
l-NAME
Cumulative addition of PE (1 nM10 AM) to organ bath
resulted in concentration-dependent contraction of aorta of all
groups (Fig. 1). Maximal contraction (E
max
, g) and pD
2
value in case of diabetic control (1.69T0.45, 7.00T0.040)
were significantly ( p <0.05) higher as compared to nondia-
betic control (1.48T0.31, 6.63T0.023) and metformin treat-
ment to diabetic animals (1.51 T0.42, 6.64 T0.033)
significantly reduced it. The presence of l-NAME signifi-
cantly ( p <0.05) increased the contractile response to PE in
aortic rings of nondiabetic control, diabetic control and D-
MET groups. There was no significant change in PE-induced
contraction in diabetic control group due to the presence of
l-NAME (Fig. 1).
Relaxation response to Ach and SNP on aorta obtained from
control and metformin-treated rats
Addition of Ach in all aortic rings with intact endothelium
resulted in concentration-dependent relaxation of rings that
were precontracted with PE (Fig. 2). Percentage relaxation
and pD
2
value to Ach in aorta obtained from diabetic control
group (45.6 T4.25 and 6.95 T0.065) was significantly
( p <0.05) reduced as compared to nondiabetic control
(96.3T4.56 and 7.11T0.070) and metformin treatment to
diabetic rats (81.7T2.73 and 7.13T0.071) significantly
improved it. Addition of SNP completely relaxed aortic
rings of all the groups and there was no significant change to
SNP-induced relaxation in any of the groups (data not
shown).
Effects of indomethacin, l-NAME and methylene blue on Ach-
induced endothelium-dependent relaxation on aorta obtained
from control and metformin-treated rats
Ach completely relaxed precontracted aortic rings in
both nondiabetic groups (control and metformin-treated)
-6.5 -6.0 -5.5 -5.0 -4.5 -4.0 -3.5
0
10
20
30
N
D
N-MET
D-MET
*
*
*
a
Log[L-NAME](M)
%

C
o
n
t
r
a
c
t
i
o
n
N D N-MET D-MET
0
5
10
15
#
*
b

M

/

g

p
r
o
t
e
i
n

Fig. 4. (a) Percent contraction of endothelium intact aortic rings to different
concentration of l-NAME. The aortic rings obtained from N ( g), D ( ?),
N-MET ( >) and D-MET ( g) groups were moderately contracted with
phenylephrine before obtaining cumulative responses to l-NAME. (b) Aortic
nitrite levels determined from N, D, N-MET and D-MET groups. Values are
expressed as meanTS.E.M. *p <0.05, compared to D;
#
p <0.05, compared to N
(N=Normal, D=Diabetic).
Table 2
Superoxide dismutase, catalase, reduced glutathione and lipid peroxidation
levels in liver, kidney and aorta of nondiabetic control (N), diabetic control (D),
nondiabetic animals treated with metformin (N-MET) and diabetic animals
treated with metformin (D-MET)
Groups Liver Kidney Aorta
Superoxide dismutase
(Unit/mg protein)
N 8.23T0.241 8.83T0.056 6.19T0.118
D 4.67T0.383
a
5.37T0.135
a
4.29T0.148
a
N-MET 7.97T0.176 8.74T0.087 5.82T0.167
D-MET 7.21T0.214
b
7.65T0.133
b
5.89T0.154
b
Catalase (AM of H
2
O
2
consumed /(min mg protein)
N 11.36T0.47 11.43T0.49 5.88T0.064
D 7.73T0.415
a
7.25T0.63
a
3.64T0.136
a
N-MET 11.80T0.412 11.74T0.25 5.69T0.042
D-MET 9.83T0.812
b
9.76T0.34
b
5.56T 0.195
b
Reduced glutathione
(Ag of GSH/mg protein)
N 9.86T0.107 11.53T0.054 2.83T0.078
D 4.65T0.047
a
4.34T0.132
a
0.83T0.024
a
N-MET 9.56T0.112 11.18T0.125 2.42T0.073
D-MET 7.84T0.214
b
8.53T0.367
b
1.65T0.086
b
Lipid peroxidation
(nM of MDA/mg protein)
N 0.786T0.036 0.853T0.034 0.221T0.011
D 1.458T0.116
a
1.783T0.122
a
0.585T0.062
a
N-MET 0.790T0.069 0.971T0.056 0.219T0.086
D-MET 0.978T0.138
b
0.912T0.157
b
0.307T0.059
b
Values are expressed as meanTS.E.M. (n =67).
a
p <0.001, compared to nondiabetic control group.
b
p <0.01, compared to diabetic control.
J.B. Majithiya, R. Balaraman / Life Sciences 78 (2006) 26152624 2619
and Ach-induced relaxation was completely blocked due
to presence of l-NAME or methylene blue (Fig. 3a,b).
On the other hand, the presence of indomethacin
decreased Ach-induced relaxation in both nondiabetic
groups. The decreased Ach-induced relaxation in diabetic
control group was significantly increased by metformin
treatment. Ach-induced relaxation in diabetic groups (control
and metformin-treated) were unaltered due to presence of
indomethacin while Ach-induced relaxation was completely
blocked due to presence of l-NAME or methylene blue
(Fig. 3c,d).
Basal nitric oxide release and aortic nitrite levels
Addition of l-NAME in aortic preparation resulted in
increasing contraction of the aorta of all the groups.
Contraction was significantly higher in metformin-treated
diabetic group as compared to diabetic control group
(Fig. 4a). Aortic nitrite levels of various groups are shown
in Fig. 4b. Aortic nitrite levels were significantly ( p <0.05)
higher in diabetic control group as compared to nondia-
beticcontrol group. Metformin treatment significantly
( p <0.05) reduced aortic nitrite of diabetic animals as
compared to diabetic control group. There was no significant
change on aortic nitrite levels due to metformin treatment in
nondiabetic animals as compared to nondiabetic control
group.
Superoxide dismutase, catalase, reduced glutathione and lipid
peroxidation
Oxidative stress was significantly ( p <0.001) increased
in liver, kidney and aorta of diabetic control group
as compared to nondiabetic control group (Table 2).
SOD, CAT and GSH were significantly decreased while
lipid peroxidation was significantly increased in diabetic
control group. Metformin treatment significantly ( p <0.01)
increased levels of endogenous antioxidants (SOD, CAT
and GSH) in liver, kidney and aorta as compared to dia-
betic control (Table 2). Moreover, lipid peroxidation was
also significantly ( p <0.01) decreased in liver, kidney and
aorta of diabetic animals treated with metformin as
0.0
0.5
1.0
1.5
2.0
a
vehicle




1 mM
10 mM
*
*
-9 -8 -7 -6 -5
0.0
0.5
1.0
1.5
2.0
*
*
b
log [PE](M)
T
e
n
s
i
o
n

(
g
)
0.1 M
1 M
10 M
100 M
T
e
n
s
i
o
n

(
g
)
Fig. 5. Concentration response curve of phenylephrine on aortic rings obtained
from untreated age matched non diabetic rats (a) and STZ diabetic rats (b) in
presence of varying concentration of metformin (0.1 AM10 mM). Values are
expressed as meanTS.E.M. *p <0.05, compared to vehicle.
0
20
40
60
80
100
120
a
vehicle


1 mM
10 mM
0.1 M
-10 -9 -8 -7 -6 -5
0
20
40
60
80
100
120
b
*
*
*
log [Ach] (M)

%

R
e
l
a
x
a
t
i
o
n

%

R
e
l
a
x
a
t
i
o
n
1 M
10 M
100 M
Fig. 6. Concentration dependent relaxation of acetylcholine on aortic rings
(with intact endothelium) of untreated age matched non diabetic rats (a) and
STZ diabetic rats (b), precontracted with PE and in presence of varying
concentration of metformin (0.1 AM10 mM). Tension is expressed as
percentage relaxation of initial response to PE. Values are expressed as
meanTS.E.M. *p <0.05, compared to vehicle.
J.B. Majithiya, R. Balaraman / Life Sciences 78 (2006) 26152624 2620
compared to diabetic control. There was no significant
change in SOD, CAT, GSH and lipid peroxidation of
nondiabetic groups.
Effect of presence of metformin on concentrationresponse
curve of PE, Ach and SNP in aorta obtained from untreated
nondiabetic and STZ-diabetic rats
The presence of metformin (0.1 AM100 AM) had no
significant effect on contractile effect of PE on aorta of
untreated nondiabetic and diabetic rats (Fig. 5). On the other
hand, the presence of higher concentration of metformin (1
mM and 10 mM) significantly ( p <0.05) decreased the
contractile effect of PE. Metformin caused a concentration-
dependent rightward shift on the contractile response of PE.
Ach completely relaxed aorta obtained from nondiabetic rats
but relaxation was impaired in aortic rings from diabetic rats.
There was no significant change in relaxation response to Ach
in aorta of nondiabetic rats due to the presence of metformin
(Fig. 6). Presence of lower concentration of metformin (0.1
AM10 AM) had no significant effect on relaxation response to
Ach in aorta of diabetic rats, but the presence of higher
concentration of metformin (100 AM and higher) significantly
( p <0.05) increased relaxation (Fig. 6). SNP-induced relaxa-
tion in case of nondiabetic rats (data not shown) and diabetic
rats (Fig. 7) were similar and there was no significant effect of
-13 -12 -11 -10 -9 -8 -7 -6 -5 -4
0
20
40
60
80
100
120
Vehicle
0.1 M

10 mM
log [SNP] (M)
%

R
e
l
a
x
a
t
i
o
n
1 M
10 M
100 M
1 M
Fig. 7. Concentration dependent relaxation of sodium nitroprusside on
endothelium denuded aortic rings of untreated age matched STZ diabetic rats
precontracted with PE and in presence of varying concentration of metformin
(0.1 AM10 mM). Tension is expressed as percentage relaxation of initial
response to PE. Values are expressed as meanTS.E.M.
0
25
50
75
100
125
a

%
R
e
l
a
x
a
t
i
o
n

-9 -8 -7 -6 -5 -4 -3 -2 -1 0
0
20
40
60
80
100
120
PE + E
PE + E + L NAME
PE - E
b
log [Metformin] (M)

%
R
e
l
a
x
a
t
i
o
n

Fig. 8. Concentration-dependent relaxation of metformin on untreated age
matched non diabetic rats (a) and STZ diabetic rats (b), precontracted with PE
with intact endothelium (+E), PE with denuded endothelium (E), PE with
intact endothelium (+E) in presence of 100 M l-NAME. Values are expressed
as meanTS.E.M.
0
25
50
75
100
125
a

%

R
e
l
a
x
a
t
i
o
n
-9 -8 -7 -6 -5 -4 -3 -2 -1 0
0
20
40
60
80
100
120
b
PE + Glybenclamide
PE + TEA + 4AP
PE + TEA
PE + 4AP
PE + Ba
+
PE + vehicle
log [Metformin] (M)

%

R
e
l
a
x
a
t
i
o
n
Fig. 9. Concentration dependent relaxation of metformin on untreated age
matched non diabetic rats (a) and STZ diabetic rats (b), precontracted with PE
( n) with intact endothelium, in presence of 1 mM of TEA ( >), 100 AM
of Ba
+
( g), 1 mM of 4-AP ( ?), 10 AM of Glybenclamide ( q) and (1
mM) TEA+ (1 mM) 4-AP ( r). Values are expressed as meanTS.E.M.
J.B. Majithiya, R. Balaraman / Life Sciences 78 (2006) 26152624 2621
SNP-induced relaxation due the presence of metformin (0.1
AM10 mM) as compared to vehicle.
Relaxation response to metformin on precontracted aorta
obtained from untreated nondiabetic and STZ-diabetic rats
Addition of metformin (10 nM10 mM) to PE contracted
rings produced concentration-dependent relaxation in nondia-
betic and diabetic rats (Fig. 8). Presence of l-NAME
completely blocked metformin-induced relaxation at lower
concentration (10 nM30 M), but not at concentrations at
above 30 M. Removal of endothelium blocked metformin-
induced relaxation at concentration lower than 30 M but not
above 30 M. Further based on our observations that depolar-
ization with KCl attenuated the potency of metformin, we
attempted to determine the contribution of K
+
channels to
metformin-mediated relaxation. There was no significant effect
on metformin-mediated relaxation due to the presence of
barium ion or glybenclamide (Fig. 9). While metformin-
mediated relaxation was partially blocked due to the presence
of 4-aminopyridine and TEA, combination of 4-aminopyridine
and TEA further blocked metformin-induced relaxation.
Discussion
The blood pressure of 8-week STZ-diabetic rats was
significantly higher as compared to nondiabetic control. The
results are in concurrence with our previous study where the
blood pressure was increased after 8 weeks of STZ adminis-
tration and Ach-induced relaxation was impaired in aortic rings
(Majithiya et al., 2005). Administration of metformin for 4
weeks restored the elevated blood pressure, reduced the
enhanced contractibility to PE- and Ach-induced relaxation
was restored. In metformin-treated STZ-diabetic rats, there was
an increase in Ach-induced relaxation which may be due to
involvement of NO pathway since the relaxation was blocked
in the presence of l-NAME and not in the presence of
indomethacin. Moreover, relaxation to Ach was also blocked in
the presence of cGMP blocker, methylene blue, suggesting a
role of cGMP in elevated relaxation to Ach in STZ-diabetic
aorta. Further, tone-related basal nitric oxide studies showed
that metformin treatment significantly increased the basal nitric
oxide release in aortas of STZ-diabetic rats. This is in
accordance with the study of Sartoretto et al. (2005), where it
was shown that metformin treatment increases nitric oxide
activity but not expression and improves microvascular
reactivity in n- STZ-diabetic rats.
Metformin is reported to lower blood pressure in various
animal modelsfructose-fed rats (Verma et al., 1994), OLETF
rats (Kosegawa et al., 1996), SHR (Bhalla et al., 1996; Muntzel
et al., 1999) and insulin-resistant rats (Katakam et al., 2000). In
this study, metformin had no significant effect on blood
glucose levels in nondiabetic and diabetic rats. In the present
study, lower dose of metformin (150 mg/kg) was used and it
did not produce any significant effect on blood glucose level
Metformin reduces blood glucose level at a much higher dose
of 450500 mg/kg in STZ-diabetic rats (Stepensky et al.,
2002). Hence, the results produced are independent of
metformin effect on blood glucose level. Recently metformin
is reported to improve microvascular function in type 2 diabetic
model without improving hyperglycemia (Sartoretto et al.,
2005). Metformin is reported to reduce blood pressure by a-
adrenergic blockade or ganglionic blockade (Muntzel et al.,
1997), by inhibiting sympathetic tone (Petersen and Dibona,
1996), by direct effect on vascular smooth muscle (Sharma and
Bhalla, 1995; Peuler et al., 1997; Verma et al., 1996) or by
opening of voltage-dependent potassium channels (K
v
)
(Mather et al., 2001).
In the present study, in vitro experiments were carried out to
investigate the direct effect of metformin on aorta of untreated
nondiabetic and STZ-diabetic rats. PE-induced contraction and
Ach-induced relaxation studies in the presence of various
concentration of metformin showed that low concentration of
this drug did not have any effect on the PE-induced contraction
or Ach-induced relaxation. But presence of higher concentra-
tion (greater than 1 mM) of metformin caused significant
changes in doseresponse curves of PE and Ach. Further
metformin-induced relaxation in rings with intact endothelium
was blocked in the presence of l-NAME at lower concentra-
tions but not at higher concentrations. The effect of metformin-
induced relaxation in the presence of various potassium
channel inhibitors shows that the vasorelaxation induced by
metformin can be attributed in part to the opening of K
v
and
K
Ca+
channels. Further there was no relaxation at lower
concentration, relaxation increased only above 10 M. Similarly
significant change in PE-induced contraction and Ach-induced
relaxation was present at higher concentration (greater than 1
mM). Pharmacokinetic studies by Stepensky et al. (2002) have
shown that concentration close to 120 M was found in blood
after administering 500 mg/kg of metformin orally. In the
present study much lower dose of metformin was administered,
hence blood pressure lowering effect cannot be completely
attributed to direct effect of metformin on vascular smooth
muscle. Treatment with metformin caused reduction in
oxidative stress; it may be one of the reasons of decrease in
blood pressure coupled with restored endothelium function of
STZ-diabetic rats.
Nitric oxide is rapidly inactivated by O
2

and it has been

reported that an enhanced formation of O
2

radical may be
involved in the accelerated break down of nitric oxide
(Gryglewski et al., 1986; Rubanyi and Vanhoutte, 1986).
Moreover it has been shown that rapid destruction of nitric
oxide occurs in streptozotocin-induced diabetic rats (Kamata
and Kobayashi, 1996). The protective effect of metformin
against oxidative stress may prevent the breakdown of nitric
oxide, which may improve vascular function. Similar observa-
tions have reported that metformin reduce oxidative stress in
various animal models (Bonnefont-Rousselot et al., 2003;
Srinivasan and Carani, 2002; Faure et al., 1999). Metformin is
also reported to modulate nitric oxide synthase (Kumar et al.,
2001) and restore endothelial function (Mather et al., 2001).
Moreover recently metformin is reported to increase nitric
oxide activity without increasing nitric oxide expression in type
2 diabetic n-STZ model (Sartoretto et al., 2005). This increase
J.B. Majithiya, R. Balaraman / Life Sciences 78 (2006) 26152624 2622
in nitric oxide activity may be due to the reduction in oxidative
stress by metformin. Zou et al. (2004) have shown that
metformins action is mediated by the activation of AMP-
activated protein kinase via mitochondrial reactive nitrogen
species. On the contrary, Gallo et al. (2005) have recently
shown that metformin stimulates AMPK on one site, whereas it
inhibits PKC on the other. Gallo et al. (2005) has also shown
that metformin action involves its antioxidant effect. Our study
is in accordance with Gallo et al. (2005), showing the
antioxidant effect of metformin. Hence, the restored endothelial
function could also be attributed to the protective effect of
metformin against oxidative stress.
Hence, the restored endothelial function along with direct
effect of metformin on aortic rings and protective effect of
metformin against oxidative stress altogether may attribute to
metformins beneficial effect in STZ-diabetic rats.
Acknowledgement
Financial assistance provided by The M.S. University of
Baroda to Mr. Jayesh B Majithiya is highly acknowledged.
References
Aebi, H., 1984. Oxidoreductases acting on groups other than CHOH: catalase.
In: Colowick, S.P., Kaplan, N.O., Packer, L. (Eds.), Methods in Enzymol-
ogy. Academic Press, London, pp. 121125.
Bafna, P.A., Balaraman, R., 2004. Anti-ulcer and antioxidant activity of DHC-
1, a herbal formulation. Journal of Ethnopharmacology 90, 123127.
Bhalla, R.C., Toth, K.F., Tan, E., Bhatty, R.A., Mathias, E., Sharma, R.V., 1996.
Vascular effects of metformin: possible mechanisms for its antihypertensive
action in the spontaneously hypertensive rat. American Journal of
Hypertension 9, 570576.
Bonnefont-Rousselot, D., Raji, B., Walrand, S., GardeS-Albert, M., Jore, D.,
Legrand, A., Peynet, J., Vasson, M.P., 2003. An intracellular modulation of
free radical production could contribute to the beneficial effects of
metformin towards oxidative stress. Metabolism 52, 586589.
Calle-Pascual, A.L., Garcia-Honduvilla, J., Martin-Alvarez, P.J., Vara Calle,
J.R., Munguira, M.E., Maranes, J.P., 1995. Comparison between
acarbose, metformin, and insulin treatment in type 2 diabetic patients
with secondary failure to sulfonylurea treatment. Diabetes & Metabolism
21, 256260.
Campbell, I.W., Duncan, C., Patton, N.W., Broadhead, T., Tucker, G.T., Woods,
H.F., 1987. The effect of metformin on glycaemic control, intermediary
metabolism and blood pressure in non-insulin-dependent diabetes mellitus.
Diabetes & Metabolism 4, 337341.
Faure, P., Rossini, E., Wiernspeger, N., Richard, M.J., Favier, A., Halimi, S.,
1999. An insulin sensitizer improves the free radical defense system
potential and insulin sensitivity in high fructose-fed rats. Diabetes 48,
353357.
Gallo, A., Ceolotto, G., Pinton, P., Iori, E., Murphy, E., Rutter, G.A., Rizzuto,
R., Semplicini, A., Avogaro, A., 2005. Metformin prevents glucose-induced
protein kinase c-h2 activation in human umbilical vein endothelial cells
through an antioxidant mechanism. Diabetes 54, 11231131.
Giugliano, D., Acampora, R., De Rosa, N., Buoninconti, R., Di Maro, G.,
Donofrio, F., Marfella, R., 1993. Metformin improves glucose lipid
metabolism, and reduces blood pressure in hypertensive, obese women.
Diabetes Care 16, 13871390.
Gryglewski, R.J., Palmer, R.M.J., Moncada, S., 1986. Superoxide anion is
involved in the breakdown of endothelium-derived vascular relaxing factor.
Nature 320, 454456.
GudbjoRnsdottir, S., Friberg, P., Elam, M., Attvall, S., LoNnroth, P., Wallin,
G., 1994. The effect of metformin and insulin on sympathetic nerve activity,
norepinephrine spillover and blood pressure in obese, insulin resistant,
normoglycemic, hypertensive men. Blood Pressure 3, 394403.
Guevara, I., Iwanejko, J., Dembinska-Kiec, A., Pankiewicz, J., Wanat, A.,
Anna, P., Golabek, I., Bartus, S., Malczewska-Malec, M., Szczudlik, A.,
1998. Determination of nitrite/nitrate in human biological material by the
simple Griess reaction. Clinica Chimica Acta 274, 177188.
Hayashi, T., Fukuto, J.M., Ignarro, L.J., Chaudhuri, G., 1992. Basal release of
nitric oxide from aortic rings is greater in female rabbits than in male
rabbits: implications for atherosclerosis. Proceedings of the National
Academy of Sciences of the United States of America 89, 1125911263.
Kamata, K., Kobayashi, T., 1996. Changes in superoxide dismutase mRNA
expression by streptozotocin-induced diabetes. British Journal of Pharma-
cology 119, 583589.
Kamata, K., Miyata, N., Kasuya, Y., 1989. Impairment of endothelium-
dependent relaxation and changes on levels of cyclic GMP in aorta from
streptozotocin-induced diabetic rats. British Journal of Pharmacology 97,
614618.
Katakam, P.V.G., Ujhelyi, M.R., Hoenig, M., Miller, A.W., 2000. Metformin
improves vascular function in insulin-resistant rats. Hypertension 35,
108112.
Kobayashi, T., Kamata, K., 1999. Relationship among cholesterol, superoxide
anion and endothelium-dependent relaxation in diabetic rats. European
Journal of Pharmacology 367, 213222.
Kosegawa, I., Katayama, S., Kikuchi, C., Kashiwabara, H., Negishi, K., Ishii,
J., Inukai, K., Oka, Y., 1996. Metformin decreases blood pressure and
obesity in OLETF rats via improvement of insulin resistance. Hypertension
Research 19, 3741.
Kumar, V.B., Bernardo, A.E., Vyas, K., Franko, M., Farr, S., Lakshmanan, L.,
Buddhiraju, C., Morley, J.E., 2001. Effect of metformin on nitric oxide
synthase in genetically obese (ob/ob) mice. Life Sciences 69, 27892799.
Landin, K., Tengborn, I., Smith, U., 1991. Treating insulin resistance in
hypertension with metformin reduces both blood pressure and metabolic
risk factors. Journal of Internal Medicine 229, 181187.
Majithiya, J.B., Parmar, A.N., Balaraman, R., 2005. Pioglitazone, a PPAR g
agonist, restores endothelial function in aorta of streptozotocin-induced
diabetic rats. Cardiovascular Research 66, 150161.
Mather, K.J., Verma, S., Anderson, T.J., 2001. Improved endothelial function
with metformin in type 2 diabetes mellitus. Journal of the American College
of Cardiology 37, 13441350.
Matthaei, S., Stumvoll, M., Kellerer, M., Haring, H., 2000. Pathophysiology
and pharmacological treatment of insulin resistance. Endocrine Reviews 21,
585618.
Mishra, H.P., Fridovich, I., 1972. The role of superoxide anion in the auto-
oxidation of epinephrine and a simple assay for superoxide dismutase.
Journal of Biological Chemistry 247, 31703175.
Moller, D.E., 2001. New drug targets for type 2 diabetes and the metabolic
syndrome. Nature 414, 821827.
Moron, M.S., Depierre, J.W., Mannervik, B., 1979. Levels of glutathione,
glutathione reductase and glutathione S-transferase activities in rat lung and
liver. Biochimca and Biophysica Acta 582, 6778.
Muntzel, M.S., Abe, A., Petersen, J.S., 1997. Effects of adrenergic, cholinergic,
and ganglionic blockade on acute depressor responses to metformin in
spontaneously hypertensive rats. Journal of Pharmacology and Experimen-
tal Therapeutics 281, 618623.
Muntzel, M.S., Hamidou, I., Barrett, S., 1999. Metformin attenuates salt-
induced hypertension in spontaneously hypertensive rats. Hypertension 33,
11351140.
Oyama, Y., Kawasaki, H., Hattori, Y., Kanno, M., 1986. Attenuation of
endothelium-dependent relaxation in aorta from diabetic rats. European
Journal of Pharmacology 131, 7578.
Petersen, J.S., Dibona, G.F., 1996. Acute sympathoinhibitory actions of
metformin in spontaneously hypertensive rats. Hypertension 27, 619625.
Peuler, J.D., Miller, J.A., Bourghli, M., Zammam, H.Y., Soltis, E.E., Sowers,
J.R., 1997. Disparate effects of antidiabetic drugs on arterial contraction.
Metabolism 46, 11991205.
Rubanyi, G.M., Vanhoutte, P.M., 1986. Superoxide anions and hyperoxia
inactivate endothelium-derived relaxing factor. American Journal of
Physiology 250, H822H827.
J.B. Majithiya, R. Balaraman / Life Sciences 78 (2006) 26152624 2623
Sartoretto, J.L., Melo, G.A., Carvalho, M.H., Nigro, D., Passaglia, R.T.,
Scavone, C., Cuman, R.K., Fortes, Z.B., 2005. Metformin treatment
restores the altered microvascular reactivity in neonatal streptozotocin-
induced diabetic rats increasing NOS activity, but not NOS expression. Life
Science 77 (12), 26762689.
Senses, V., Ozyazgan, S., Ince, E., Tuncdemir, M., Kaya, F., Ozturk, M.,
Sultuybek, G., Akkan, A.G., 2001. Effect of 5-aminoimidazole-4-carbox-
amide riboside (AICA-r) on isolated thoracic aorta responses in streptozo-
tocin-diabetic rats. Journal of Basic and Clinical Physiology and
Pharmacology 12, 227248.
Sharma, R.V., Bhalla, R.C., 1995. Metformin attenuates agonist-stimulated
calcium transients in vascular smooth muscle cells. Clinical and Experi-
mental Hypertension 17, 913929.
Slater, T.F., Sawyer, B.C., 1971. The stimulatory effects of carbon tetrachloride
and other halogenoalkanes or peroxidative reactions in rat liver fractions in
vitro. Biochemical Journal 123, 805814.
Srinivasan, S., Carani, V.A., 2002. Metformin improves liver antioxidant
potential in rats fed a high-fructose diet. Asia Pacific Journal of Clinical
Nutrition 11, 319322.
Stepensky, D., Friedman, M., Raz, I., Hoffman, A., 2002. Pharmacokinetic
pharmacodynamic analysis of the glucose-lowering effect of metformin in
diabetic rats reveals first-pass pharmacodynamic effect. Drug Metabolism
and Disposition 30, 861868.
Verma, S., Bhanot, S., Mcneill, J.H., 1994. Antihypertensive effects of
metformin in fructose-fed hyperinsulinemic, hypertensive rats. Journal of
Pharmacology and Experimental Therapeutics 271, 13341337.
Verma, S., Bhanot, S., Mcneill, J.H., 1996. Decreased vascular reactivity in
metformin-treated fructose-hypertensive rats. Metabolism 45, 10531055.
Zanetti, M., Sato, J., Katusic, Z.S., Obrien, T., 2001. Gene transfer of
superoxide dismutase isoforms reverses endothelial dysfunction in diabetic
rabbit aorta. American Journal of Physiology 280, H2516H2523.
Zou, M.H., Kirkpatrick, S.S., Davis, B.J., Nelson, J.S., Wiles, W.G., Schlattner,
U., Neumann, D., Brownlee, M., Freeman, M.B., Goldman, M.H., 2004.
Activation of the AMP-activated protein kinase by the anti-diabetic drug
metformin in vivo: role of mitochondrial reactive nitrogen species. Journal
of Biological Chemistry 279, 4394043951.
J.B. Majithiya, R. Balaraman / Life Sciences 78 (2006) 26152624 2624