Clinical and Experimental Pharmacology and Physiology (2015) 42, 964–971 doi: 10.1111/1440-1681.

12441

ORIGINAL ARTICLE

Activation of imidazoline-I3 receptors ameliorates pancreatic damage

Yingxiao Li,* Kai-Chun Cheng,* Akihiro Asakawa,* Haruka Amitani,* Yoshiyuki Takimoto,†
Joshua Runtuwene* and Akio Inui†
*Department of Psychosomatic Internal Medicine, Kagoshima University Graduate School of Medical and Dental
Sciences, Kagoshima, Japan and †Department of Stress Sciences and Psychosomatic Medicine, Graduate School of
Medicine, The University of Tokyo, Tokyo, Japan

SUMMARY INTRODUCTION
Diabetic disorders are associated with progressive b cell failure, and
Agmatine, an endogenous ligand of imidazoline receptors,
apoptosis is associated with b cell damage. In type 1 diabetes, the b
is reported to exhibit anti-hyperglycaemic and many other
cell mass is reduced by 70–80% at the time of diagnosis, which is
effects. It has been established that the imidazoline I3 recep-
typically caused by autoimmune assault.1 In type 2 diabetes, there is
tor is involved in insulin secretion. The current study charac-
also a significant reduction in b cell mass and a threefold increase in
terizes the role of the imidazoline I3 receptor in the
b cell apoptosis.2 The mechanisms leading to b cell death in diabetes
protection of pancreatic islets. The activity effect of agmatine
are related to cytokines, inflammatory mediators, nitric oxide (NO)
against on streptozotocin (STZ)-induced (5 mmol/L) rat b cell
and oxygen free radicals, and these mechanisms activate a final com-
apoptosis was examined by using ApoTox-Glo triplex assay,
mon pathway involving interleukin (IL)-1b, nuclear factor (NF)-jB,
live/dead cell double staining assay, flow cytometric analysis,
and Fas.3 Thus, prevention of pancreatic damage is an important
and western blot. Imidazoline I3 receptors antagonist KU14R
prophylactic measure in the control and management of hypergly-
and the phospholipase C inhibitor named U73122 were trea-
caemia.4 Development of therapeutic strategies to protect b cells
ted in b cells to investigate the potential signalling pathways.
from in diabetic patients is an important issue.5
The serum glucose and recovery of insulin secretion were
Streptozotocin (STZ), a glucose analog glucopyranose, 2-
measured in STZ-treated rats after continuously injected
deoxy-2-(3-methyl- nitrosoureido-D), induces diabetes in experi-
agmatine. The apoptosis in rat b cells was reduced by agma-
mental animals because of its relatively specific cytotoxic effects
tine in a dose-dependent manner, cell viability was improved
on b cells.6 The cytotoxic effect of STZ at low doses can cause
after treatment with agmatine and these effects were sup-
apoptosis of pancreatic b cells, whereas at higher doses of STZ,
pressed after the blockade of KU14R and U73122. Western
the predominant mode of cell death is necrosis.7 Thus, STZ is
blot analysis confirmed that agmatine could decrease caspase-
widely used in studies of diabetic disorders.
3 expression and increase the p-BAD levels. In STZ-treated
The imidazoline receptor mediates the antihypertensive action of
rats, injection of agmatine for 4 weeks may significantly
clonidine and has analogs in the brain.8 There are three main imi-
lower the serum glucose and recovery of insulin secretion.
dazoline receptor subtypes. The imidazoline 1 (I1) receptor regu-
This improvement of pancreatic islets induced by agmatine
lates the sympatho-inhibitory pathway to lower blood pressure,
was deleted by KU14R in vivo. Agmatine can activate the imi-
and the I2 receptor is an important binding site for factors involved
dazoline I3 receptor linked with the phospholipase C pathway
in metabolism.9 Induction of insulin secretion from pancreatic b
to induce cell protection against apoptosis induced by a low
cells via the binding of imidazoline receptor ligands is tentatively
dose of STZ. This finding provides new insight into the pre-
thought to be the role of the I3 receptors.10 Agmatine ((4-aminobu-
vention of early stage pancreatic islet damage.
tyl) guanidine) is the decarboxylation product of arginine and is an
Key words: agmatine, apoptosis, beta cell, imidazoline-I3
intermediate in polyamine biosynthesis.11 Agmatine may improve
receptors.
insulin sensitivity and produce a hypoglycaemic effect after bind-
ing to the imidazoline receptor.12
Recently, incretin has been shown to induce insulin secretion
and protect pancreatic b cells.13 Similar to incretin, agmatine has
the ability to induce insulin secretion. Agmatine has also been
mentioned to activate I3 receptors located in pancreatic tissue. It
Correspondence: Akio Inui, Department of Psychosomatic Internal
is of special interest to know whether agmatine can also protect
Medicine, Kagoshima University Graduate School of Medical and Dental
Sciences, 8-35-1 Sakuragaoka, Kagoshima, Japan. Email: inui@m.kufm. pancreatic b cells. The present study aimed to identify the role of
kagoshima-u.ac.jp agmatine in pancreatic protection and investigate the relationship
Received 19 January 2015; revision 25 May 2015; accepted 9 June of agmatine with imidazoline I3 receptors both in vivo and
2015. in vitro under treatment with low-dose STZ.
© 2015 Wiley Publishing Asia Pty Ltd
 Imidazoline-I3 ameliorates pancreatic damage
RESULTS
Agmatine attenuated the streptozocin-induced cytotoxicity
and apoptosis in b cells
Exposure of b cells to 5 mmol/L STZ for 6 h significantly
induced apoptosis in a medium containing 25 mmol/L glucose.
To further evaluate the protective role of agmatine against
streptozocin in vitro, b cell viability/cytotoxicity and apoptosis
was measured using an ApoTox-Glo Triplex Assay (Promega,
Madison, WI, USA). The STZ treatment resulted in a signifi-
cant reduction in the number of viable b cells, and cell toxic-
ity was significantly increased compared with the control. In
contrast, treatment with agmatine at different concentrations (5,
10 and 100 lmol/L) caused a dose-dependent reduction in the
apoptosis induced by STZ, thereby increasing cell viability and
decreasing cytotoxicity. These results indicate that agmatine
reduced STZ-induced cell toxicity in a dose-dependent manner
(Fig. 1a–c).
Agmatine increased the viability of STZ-treated b cells
The viability of b cells was assayed using the Live/Dead assay
kit, which differentially stains living and dead cells. Living cells
were detected upon excitation at 488 nm (green emission), and
dead cells were detected at 532 nm (red emission). In Fig. 2,
pre-treatment with 5 mmol/L STZ for 6 h resulted in a higher
proportion of b cells with EthD-1 positive nuclei. The images
(a)
(b)
(c)
Fig. 1 ApoTox-Glo Triplex assay showing the viability, cytotoxicity,
and apoptosis of rat b islets treated with 5 mmol/L streptozotocin,
5 mmol/L STZ + 5 lmol/L agmatine, 5 mmol/L STZ + 10 lmol/L agma-
tine, and 5 mmol/L STZ + 100 lmol/L agmatine. The data are presented
as the mean  SEM; n = 3. *Significantly different from the control.
© 2015 Wiley Publishing Asia Pty Ltd
965
indicate that STZ induced cell death when compared with the
control group. Adding 5 lmol/L agmatine for 30 min signifi-
cantly decreased cell death compared with the vehicle-treated
group. KU14R is an antagonist of the atypical imidazoline bind-
ing site (putative I3 receptor) of pancreatic b cells, which selec-
tively blocks efaroxan-induced insulin secretion. Therefore,
treatment with 1 mmol/L KU14R for 30 min resulted in a signifi-
cant loss of agmatine positivity and a large increase in EthD-1
incorporation.
Agmatine decreased the percentage of apoptotic b cells in
flow cytometric analysis
As shown in flow cytometric analysis, the majority of cells in
the negative control were localized in the lower left quadrant,
indicating the presence of viable cells without apoptotic stimula-
tion. When the cells were treated with STZ for 6 h, a certain pro-
portion of the cells were observed in the lower right quadrant
(58.3%), indicating the presence of apoptotic cells. Upon treat-
ment with agmatine, there was a shift in the proportion from the
lower right quadrant to the lower left quadrant. The percentage of
apoptotic cells decreased to 34.8% after the cells were treated
with agmatine. However, co-treatment of cells with agmatine and
KU14R resulted in a 57.1% increase in the percentage of apop-
totic cells induced by STZ (Fig. 3).
Agmatine decreased the levels of apoptosis-related proteins
via the imidazoline I3 receptor in b cells
Caspase 3 and p-BAD are major regulatory proteins associated
with the network of apoptosis. Western blot analysis was applied
Fig. 2 Images of rat b cells treated with agmatine showing its effects on
STZ-induced apoptosis. b cells (1 9 105) were treated with 5 mmol/L
STZ, loaded with 1 lmol/L calcein-AM and excited with light at a wave-
length of 488 nm (green emission) to show viable cells. The same cells
were loaded with 1 lmol/L 4 lmol/L EthD-1 and excited with 532 nm
light (red emission) to show potential dead cells. n = 3. Scale bars,
200 lm. The cells were co-stained with the Live/Dead viability/cytotoxic-
ity assay kit for animal cells. n = 3; Scale bars, 200 lm.
966 Y Li et al.

to detect the activation of caspase 3 protein and the level of the

anti-apoptotic protein p-BAD. As shown in Fig. 4a, the expression
level of caspase 3 was increased after treatment with 5 mmol/L
STZ for 6 h (0.866  0.069) compared with the negative control
(0.564  0.034). When agmatine was added, the increased expres-
sion of caspase 3 was suppressed (0.596  0.049). Additionally,
this effect of agmatine was attenuated by KU14R at a dose suffi-
cient to block imidazoline I3 receptors (0.854  0.060). Western
blot analysis showed a significant decrease in p-BAD in b cells
that were treated with STZ alone (0.472  0.048) compared with
the negative control (0.741  0.037). In the presence of agmatine,
the p-BAD level was markedly increased (0.716  0.040), and
this increase was blocked by KU14R (0.481  0.046; Fig. 4b).
Phospholipase C is involved in an agmatine-induced cell
protective effect
U73122 is a phospholipase C (PLC) inhibitor. To understand the
role of PLC in the effects induced by agmatine, pancreatic cells
were divided into four groups. The first group was left untreated
(untreated control). The second group was treated with STZ
(5 mmol/L) for 6 h (STZ control). The third group was treated
with agmatine (5 lmol/L) for 30 min before exposure to STZ
(5 mmol/L) for 6 h. The viability/cytotoxicity and apoptosis in b
cells were measured using an ApoTox-Glo assay. Treatment of b
cells with U73122 led to an attenuation of the agmatine-induced
protective effect (Fig. 5).

Fig. 3 Flow cytometric analysis of apoptotic cells. Cells treated with 5 mmol/L STZ in the presence or absence of 5 lmol/L agmatine and 1 mmol/L
KU14R were incubated with FITC-labeled Annexin-V and propidium iodide. After washing, the cells were submitted to flow cytometric analysis. Num-
bers at the corners represent the percentage of cells found in each quadrant. All three experiments were performed in triplicate, and each figure is repre-
sentative of three independent trials, n = 3.

© 2015 Wiley Publishing Asia Pty Ltd

 Imidazoline-I3 ameliorates pancreatic damage
Fig. 4 Western blot analysis of the expression level of apoptosis-related
proteins. Cells without STZ treatment were used as a negative control
(lanes 1 and 3 in (a) and (b)). Cells treated with 5 mmol/L STZ were
subsequently treated with agmatine (lane 2) or with agmatine and KU14R
(lane 4). Total cellular proteins were collected after 6 h of treatment. The
cytosolic fraction of the cells was prepared as described in Methods and
Materials. The expression levels of caspase-3, p-BAD and b-actin were
detected by Western blot analysis. The figure is representative of three
independent trials; n = 3.
Serum glucose levels are altered in rats treated with STZ,
agmatine and KU14R
Administration of STZ led to an increase in the blood glucose
level. Rats treated with STZ and 10 mg/kg agmatine had a lower
blood glucose level. However, combined treatment with 8 mg/kg
KU14R blocked the action of 10 mg/kg agmatine in STZ-treated
rats based on changes in the blood glucose level during a 4-week
period (Fig. 6).
Effect of agmatine and KU14R on insulin secretion in STZ-
treated rats
Insulin secretion and blood glucose in STZ rats were both altered.
Plasma insulin was markedly reduced in STZ-treated rats; how-
ever, this reduction was reversed by treatment with agmatine at
10 mg/kg per day. Meanwhile, in the group treated with KU14R
(8 mg/kg per day) and agmatine (10 mg/kg per day), the plasma
insulin level was attenuated during a 4-week period (Fig. 7).
© 2015 Wiley Publishing Asia Pty Ltd
967
(a)
(b)
(c)
Fig. 5 ApoTox-Glo Triplex assay showing the (a) viability, (b) cytotoxi-
city, and (c) apoptosis of rat b cells treated with 5 mmol/L streptozotocin,
5 mmol/L STZ + 5 lmol/L agmatine, and 5 mmol/L STZ + 5 lmol/L
agmatine + 1 lmol/L U73122. The data are presented as the
mean  SEM; n = 3. *Significantly different from the control.
DISCUSSION
The protective reaction of agmatine has been proved in cerebral
ischemic injury14 and in cultured neurons and PC12 cells.15 To
investigate the role of agmatine in early stage pancreatic tissue
damage, apoptosis was induced in rat pancreatic b cells by treat-
ing them with low concentrations (5 mmol/L) of STZ for 6 h.
The data presented here demonstrate that agmatine is able to
improve the survival rate of primary b cells and protect pancre-
atic function by activation of the imidazoline I3 receptor. This
study specifically shows that the imidazoline I3 receptor has a
role in preventing STZ-induced apoptosis of pancreatic b cells.
Streptozotocin action in b cells is accompanied by characteristic
alterations in blood insulin and glucose concentrations. STZ is
taken up by pancreatic b cells via the glucose transporter 2
(GLUT2).16 The cytotoxic effect of STZ at low doses is involved
in the activation of the apoptotic pathway, in addition, predominant
b cell death occurs via necrosis at high dose of STZ treatment.7,17
Because STZ is a nitric oxide (NO) donor and NO was contributes
to STZ-induced DNA damage,7 STZ was found to generate reac-
tive oxygen species, induce DNA fragmentation, and evoke other
deleterious changes in the cells.17 Moreover, high glucose concen-
tration-induced apoptosis involves high glucose-activated NFkB,
mitochondrial cytochrome C mediated caspase 3 activation and the
formation of reactive oxygen species (ROS).18 Additionally, high
glucose could increase pancreatic cell vulnerability to toxic
968
Fig. 6 Comparison of the effect of the agmatine and the imidazoline I3
receptor inhibitor KU14R on serum glucose levels in STZ-treated rats.
Serum glucose levels were measured each day for 8 weeks. STZ-treated
rats subsequently treated with agmatine (10 mg/kg, I.P.) (white dots) and
KU14R (8 mg/kg, I.V.) (black triangles) were compared with STZ group
(black dots). Normal control as baseline was labelled as black squares.
Blood glucose levels were measured 8 days after the administration of
reagents. The data are presented as the mean  SEM; n = 8. *Significantly
different from the control. ( ), control n = 8; ( ), STZ 45 mg/kg
n = 8; ( ), STZ 45 mg/kg n = 8 + Agmatine 10 mg/kg n = 8; ( ),
STZ 45 mg/kg n = 8 + Agmatine 10 mg/kg n = 8 + KU14R n = 8.
damage by increasing the expression of potential autoantigens on
the cell membrane surface.19 In our experiment, we confirmed that
treatment with STZ at low doses (5 mmol/L) in a medium contain-
ing 25 mmol/L glucose for 6 h caused significant changes in cell
morphology. Treatment with agmatine at various concentrations
resulted in recovery, as evidenced by dose-dependent increases in
the number of surviving cells. These results indicate that agmatine
reduced STZ-induced cell toxicity.
Agmantine can bind to several subtypes of imidazoline recep-
tors,20 including I1 and I2 receptors in addition to the I3 receptor.
Fig. 7 Comparison of the effect of agmatine and the imidazoline I3
receptor inhibitor KU14R on insulin secretion in STZ-treated rats. Insulin
secretion was measured each day for 8 weeks. STZ rats treated with agma-
tine (10 mg/kg, I.P.) (white dots) and KU14R (8 mg/kg, I.V.) (black trian-
gles) were compared with the control group (black dots). Insulin secretion
was measured 8 days after reagent administration. The data are the
mean  SEM; n = 8. *Significantly different from the control. ( ),
STZ 45 mg/kg n = 8; ( ), STZ 45 mg/kg n = 8 + Agmatine 10 mg/
kg n = 8; ( ), STZ 45 mg/kg n = 8 + Agmatine 10 mg/kg n = 8 +
KU14R n = 8.
© 2015 Wiley Publishing Asia Pty Ltd
Y Li et al.
However, only I3 receptors located in pancreas b-cells play an
important role in regulating insulin secretion.10 Thus, although
agmantine belongs to a non-selective agonist of imidazoline recep-
tors, administration agmantine to b-cells will assure its action via
activation of I3 receptors in the main. The first described insulino-
tropic action of I3 imidazolines was the closure of ATP-sensitive
potassium channels (KATP), which leads to depolarization, calcium
influx and release of insulin.21 The existence of distinct I3 binding
proteins has also been suggested on the basis of pharmacological
experiments, which demonstrated that although the I3 agonist,
efaroxan, and the b-carboline, harmane, directly elevate cytosolic
Ca2+ and increase insulin secretion, these responses display differ-
ent characteristics.10 In the present study, KU14R, a specific
antagonist against the imidazoline I3 receptor, could inhibit the
agmatine-induced cell protective effect, suggesting that agmatine
functions through the imidazoline I3 receptor. In flow cytometric
analysis, cell viabilities were improved after treatment with agma-
tine, and this effect was suppressed after blockade of the I3 recep-
tor. Thus, agmatine could protect pancreatic cells via activation of
imidazoline I3 receptors.
Several pathways can initiate apoptosis.22 Some mitochondrial
proapoptotic factors, such as Ca2+-activated cysteine protease,
calpain, were involved in caspase 3 activation.23 BAD is a mole-
cule that activates AKT to promote cell survival by suppressing
apoptosis via subsequent modulation.24 To determine the role of
agmatine in pancrease cell apoptosis, we tested the expression
levels of caspase-3 and p-BAD in b cells. Agmatine caused a sig-
nificant decrease in caspase-3 expression and an increase in p-
BAD expression, which indicates that agmatine may block apop-
tosis and improve the survival rate.
Caspase 3 activation is predominantly mediated via PLC acti-
vation, as evidenced by the decrease in caspase 3 activities in the
presence of U73122.25 In b cells, high concentrations of glucose
stimulate insulin release, and increase glucose sensitivity, electri-
cal activity, and Ca2+ signalling.26 It has been demonstrated that
glucose-induced insulin secretion is dependent on ATP-regulated
K+ channels.27 Phospholipase C activation leads to the hydrolysis
of PIP2 into diacylglycerol (DAG) and IP3. Then, IP3 binds to
the IP3 receptor on endoplasmic reticulum (ER), resulting in an
increase of Ca2+ release from ER. Also, DAG will activate pro-
tein kinase C (PKC). This pathway raises the cytoplasmic free
Ca2+ concentration to enhance insulin secretion.27 Otherwise,
PKC may activate Raf1 by seperating it from Raf kinase inhibi-
tory protein (RKIP).28 Raf1 will phosphorylate and activate Mito-
gen-activated protein kinase kinase or MAPKK (MEK 1 and
MEK 2) to activate Mitogen-activated protein kinases or MAPK
(ERK1/2).29,30 Then, ERK stimulates p90RSK activity mediating
the phosphorylation of pro-apoptotic protein (BAD) at ser-112
and its binding to 14-3-3 protein will suppress the BAD-mediated
cell apoptosis.31,32 Therefore, in primary b cells and Min6 cell
lines, the cytosolic Ca2+ signal evoked by PLC-linked agonists
may be primarily frequency-encoded.33 Thus, we used a PLC
inhibitor (U73122) to determine whether PLC is the signal linked
to imidazoline receptors. Treatment with both U73122 and agma-
tine in primary b cells attenuated the cell survival rate compared
with the group only treated with agmatine. This result suggested
that agmatine can activate the imidazoline I3 receptor linked to
the PLC-dependent caspase 3 activation to induce cell protection
under low STZ conditions. Moreover, we also confirmed the
 Imidazoline-I3 ameliorates pancreatic damage
effectiveness of agmatine in vivo. After agmatine was adminis-
tered to STZ-treated rats for 4 weeks, blood glucose levels were
decreased and insulin secretion was increased. This change could
be reversed by KU14R. Our results demonstrated that agmatine
is able to protect b cells from cytotoxic damage and improve the
function of the pancreas both in vitro and in vivo.
Agmatine protected b cells after binding with the imidazoline
I3 receptor to induce the PLC-related pathway and decrease
apoptosis. This study is the first to demonstrate the protective
effect of agmatine against cell damage. Thus, agmatine and
related analogs may be an effective therapeutic agent for treating
early stage damage to islet function.
MATERIALS AND METHODS
Animals
Male Wistar rats (weighing 320–340 g) obtained from the
National Animal Centre (Taipei, Taiwan) were maintained in a
temperature-controlled room (25  1°C) under a 12:12 light–
dark cycle (lights on at 0600 hours). Food and water were avail-
able ad libitum. All animal procedures were performed according
to the Guide for the Care and Use of Laboratory Animals of the
National Institutes of Health. The animal experiments were
approved by the Regional Ethics Committee for Animal Research
at Chi-Mei Medical Centre (Tainan, Taiwan).
Islet isolation and primary culture
Pancreatic islets were aseptically isolated from rat pancreata
according to a previous method.34 The excised rat pancreas were
minced into small pieces and digested with collagenase (Roche,
Basel, Switzerland) (1 mg/mL) for 10 min. The collagenase was
then inactivated with two washes of Roswell Park Memorial
Institute (RPMI) 1640 medium (Thermo, Waltham, MA, USA)
containing 10% fetal calf serum (Sigma, St. Louis, MO, USA).
To ensure purity of the preparation, islets were hand-picked and
counted under an inverted microscope (Olympus, Tokyo, Japan).
Islet purity was determined by dithazone staining. After isolation,
islet purity reached nearly 98%. All isolated islets were cultured
in RPMI 1640 supplemented with penicillin (100 IU/mL), strep-
tomycin 100 mg/mL (Sigma), amphotericin B 2.5 mg/mL
(Gibco, Carlsbad, CA, USA) and 10% FBS (Biologic Industries,
Kibbutz Beit Haemek, Israel). The primary cultures were incu-
bated at 37°C with 5% CO2 for 48 h.
Treatment of cells with reagents
On day 3, after splitting the cells into 6-well plates, the medium
was removed and the cells were washed once in phosphate-buf-
fered saline (PBS). Fresh RPMI 1640 medium containing
25 mmol/L glucose was then added with low concentrations of
STZ (5 mmol/L) or with agmatine pretreatment at the desired
concentrations (Sigma). To ascertain which signaling pathway
was activated by agmatine, an antagonist of the imidazoline I3
receptor, KU14R (Santa Cruz Biotechnology, Santa Cruz, CA,
USA) or a phospholipase C (PLC) inhibitor, U-73122 (RBI, Nat-
ick, MA, USA) was added during the first 30 min. Agmatine
treatment was performed for 30 min, and STZ was applied and
© 2015 Wiley Publishing Asia Pty Ltd
969
incubated for 6 h. The cells were incubated in reagents contain-
ing RPMI 1640 media, which was removed at the specified
times, and the cells were washed once with PBS. Normal growth
medium was added, and the cells were allowed to recover for
6 h. After a recovery period, one batch of cells was fixed and
processed for morphology.
ApoTox-Glo triplex assay
The apoptotic response was measured by detection of DNA his-
tone complexes released from the nucleus to the cytosol using the
ApoTox-Glo Triplex Assay. The assay was performed to assess
viability, cytotoxicity and caspase-3/7 activation within a single
assay well35. The primary b cells (1 9 105 cells) were cultured in
96-well assay plates. Each well contained a final volume of
200 ml of RPMI 1640, which contained 10% FBS and 1% peni-
cillin/streptomycin. For the assay, 20 mL of the viability/cytotoxi-
city reagent containing agmatine (5, 10, 100 lmol/L) was applied
and incubated for 30 min; subsequently, the cells were treated
with STZ (5 mmol/L) for 6 h. To understand the relationships
among agmatine, the I3 receptor, and PLC, all the cells were pre-
treated with KU14R (0.2 mmol/L) or U73122 (0.2 mmol/L),
briefly mixed via orbital shaking (500 rpm for 30 s) and incubated
for 30 h at 37°C. Cells in the wells were treated with agmatine for
30 min, and STZ (5 mmol/L) was added for 6 h. After adding
100 lL of caspase-Glo 3/7 to all wells, the samples were briefly
mixed by orbital shaking (500 rpm for 30 s). After incubating for
30 min at room temperature, luminescence was measured using a
microplate reader to assess apoptosis. Fluorescence was measured
at 380 Ex/510 Em (viability), 485 Ex/520 Em (cytotoxicity) and
luminescencew (apoptosis).
Live/dead double staining assay
To analyze the effect of agmatine on b cell proliferation and sur-
vival, cell viability was tested using a Live/Dead assay (Invitro-
gen, Carlsbad, CA, USA) as described previously36. The Live/
Dead solution (100 lL) was added and incubated for 15 min in
an incubator at 37°C in a humidified atmosphere (20% O2) with
5% CO2. The staining solution was removed, and the samples
were viewed under an Olympus fluorescence microscope with
494 nm (green, Calcein) and 528 nm (red, EthD-1) excitation fil-
ters. Images were captured using Lucia software (Laboratory Ima-
ging, s.r.o., Praha, Czech Republic).
Annexin V/PI staining and flow cytometry analysis
Apoptotic cells were quantified by Annexin V-PI staining using
a flow cytometer according to a previously described method.37
The FITC Apoptosis Detection Kit (BD, San Diego, CA, USA)
was used. The cells were divided in to four groups: (i) treated
with 5 mmol/L STZ; (ii) treated with 5 mmol/L STZ and
5 mmol/L agmatine; (iii) treated with 5 mmol/L STZ, 5 mmol/L
agmatine and 10 mmol/L KU14R; or (iv) treated with the vehicle
(0.1% DMSO). b cells were plated in 12-well plates at a density
of 5 9 105 cells/well; each group was treated with different
reagents in complete medium for 48 h. At the end of each treat-
ment, the cells were collected and the quantitative apoptotic death
970
assay was performed by Annexin V and PI staining (Molecular
Probes) following the manufacturer’s protocol. Staining was then
analyzed immediately by flow cytometry using FACS.
Western blotting analysis
SDS-PAGE and Western-blot were carried out by standard proce-
dures. Caspase-3 and p-Bad expression levels in b cells were
determined by Western blotting analysis. The b cells were pre-
cultured with STZ (5 mmol/L) for 6 h, and agmatine (5 lmol/L)
was added with or without KU14R (1 mmol/L) or with vehicle
for 30 min. The b cells were washed in ice-cold PBS and lysed
for 15 min on ice. The protein concentration was measured by
the Bradford method (Bio-Rad, Hercules, CA, USA). Protein was
separated by SDS-PAGE and transferred onto PVDF membranes
(Expedeon, Cambridgeshire, UK). The membrane was blocked
with 5% skim milk in TBS containing 0.1% Tween 20 (TBST)
and incubated for 2 h. The membrane was then washed in TBST
and hybridized with caspase-3 and p-Bad antibodies (Santa
Cruz), which were diluted to a suitable concentration in TBST
for overnight at 4°C. Then membranes were incubated in sec-
ondary antibodies for 1 h at room temperature. Then, signals
were detected by using an ECL kit (Millipore Corporation, Biller-
ica, MA, USA). The bands densities were quantified by using a
laser densitometer.
Determination of serum glucose and insulin levels in STZ-
treated rats
Rats were injected intraperitoneally with 45 mg/kg streptozotocin
to induce pancreatic cell damage as described previously.38 Rats
were injected intraperitoneally with 45 mg/kg streptozotocin
(STZ) to induce pancreatic cell damage as described previously.
STZ-treated rats were randomly divided into three groups: group
one received an intravenous injection of 10 mg/kg per day agma-
tine; group two received an intravenous injection of 8 mg/kg per
day KU14R and 10 mg/kg per day agmatine; and group three
received the same volume of vehicle injected into the tail (control
group). After 30 min, both groups received an intraperitoneal
injection of agmatine at 10 mg/kg per day. The third group was
treated with similar injections containing vehicle only. All experi-
ments were performed for 8 weeks. Blood samples were obtained
from each rat under anesthesia. Glucose and insulin levels were
measured as described previously.39
Statistical analysis
Data are expressed as the mean  SEM from the number (n) of
each group. Statistical analysis was carried out using one-way
analysis of variance (ANOVA) to determine significance, and
Tukey’s test was used to determine significant differences
between relevant treatment groups. Statistical significance was set
at P < 0.05.
DISCLOSURE
The authors declare that there is no conflict of interest that could
be perceived as prejudicing the impartiality of the review.
© 2015 Wiley Publishing Asia Pty Ltd
Y Li et al.
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