Vol.2, No.2, 112-123 (2010)
Copyright © 2010 Openly accessible at http://www.scirp.org/journal/HEALTH/
Comparative effects of idazoxan, efaroxan, and BU 224
on insulin secretion in the rabbit: Not only interaction
with pancreatic imidazoline I2 binding sites
María José García-Barrado1, María Francisca Pastor1, María Carmen Iglesias-Osma1,
Christian Carpéné2, and Julio Moratinos1
1Departamento de Fisiología y Farmacología, Facultad de Medicina, Universidad de Salamanca, Salamanca, Spain; barrado@usal.es
2INSERM, U586 (Institut National de la Santé et de la Recherche Médicale), Unité de Recherches sur les Obésités, Université Paul
Sabatier, Institut Louis Bugnard IFR31, Toulouse, France
Received 6 November 2009; revised 1 December 2009; accepted 22 December 2009.
The nature of the binding site(s) involved in the
insulin secretory activity of imidazoline compo-
unds remains unclear. An imidazoline I2 binding
site (I2BS) has been neglected since the classic
I2 ligand, idazoxan, does not release insulin.
Using the rabbit as an appropriate model for the
study of this type of binding sites, we have tried
to re-evaluate the effects of idazoxan, the
selective I2 compound BU 224, and efaroxan on
insulin secretion. Mimicking efaroxan, idazoxan
and BU 224 potentiated insulin release from
perifused islets in the presence of 8 mM glucose.
In static incubation, insulin secretion induced
by idazoxan and BU 224 exhibited both dose
and glucose dependencies. ATP-sensitive K+
(KATP) channel blockade, though at a different
site from the SUR1 receptor, with subsequent
Ca2+ entry, mediates the insulin releasing effect
of the three ligands. However, additional MAO
independent intracellular steps in stimulus-
secretion coupling linked to PKA and PKC
activation are only involved in the effect of BU
224. Therefore, both an I2 related binding site at
the channel level shared by the three ligands
and a putative I3-intracellularly located binding
site stimulated by BU 224 would be mediating
insulin release by these compounds. In vivo
experiments reassess the abilities of idazoxan
and BU 224 to enhance glucose-induced insulin
secretion and to elicit a modest blood glucose
lowering response.
Keywords: BU 224; Efaroxan; Idazoxan; Imidazoline
Ligands; Insulin Secretion; IVGTT (Intravenous Glucose
Tolerance Test); KATP Channel; PK Activity; Rabbit
Pancreatic Islets
A number of imidazoline containing compounds have
been previously shown to induce insulin release from the
perifused pancreas or isolated islets [1, 2] and to improve
glucose tolerance in rats [3-5] and mice [6].
In accordance with the mechanisms of their insulino-
tropic effect, two groups of imidazoline compounds can
be considered: classical imidazolines, i.e., imidazoline
derivatives possessing both ATP-sensitive K+(KATP)
channel activity and a direct effect on exocytosis, like
RX871024 [7], and a new generation of compounds
without effect on KATP channels though possessing a pure
glucose-dependent insulinotropic effect like BL11282 [8].
The KATP channel consists of two subunits: a sulphony-
lurea receptor (SUR1) and a Kir6.2 subunit. Classical
imidazoline drugs bind to the transmembrane protein
Kir6.2 [9] considered to be the pore-forming subunit of
the channel whereas sulphonylureas bind to the SUR1
receptor. It is also established that the binding site for
imidazolines and the sulphonylurea receptor are not
identical since the first drugs do not displace binding
from the SUR sites [10]. Additional sites located at more
distal stages of the stimulus-secretion coupling pathway,
mediating activation of protein kinase A (PKA) and pro-
tein kinase C (PKC) have also been reported [8,11,12].
However, when trying to analyze the nature of the
binding sites or receptors involved in their insulin secre-
tory response a number of difficulties have emerged. An
I2 imidazoline binding site (I2-site) has been discarded
since the classic I2 ligand idazoxan exhibited a mild
concentration independent increase in insulin release [13],
failed to evoke any effect [14] or even blocked the re-
sponse induced by efaroxan (an 2-adrenoceptor antago-
nist) with an imidazoline structure [15]. Similarly, the
monoamine oxidase A (MAO-A) inhibitor clorgyline did
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not modify the insulin secretory response induced by the
imidazoline compound RX871024 [16]. Radioligand bind-
ing studies performed in membranes from RINm5F and
MIN6 cells, in the presence of [3H]-RX821002 (meth-
oxy-idazoxan an imidazoline 2-adrenoceptor antagonist)
showed a low affinity non-adrenergic binding site which
could be displaced by efaroxan but not by idazoxan
[18,19]. Therefore, evidence for a novel putative I3
binding site involved in the insulin secretory response is
being accepted. Efaroxan is tentatively considered an I3
ligand and 2-(2-ethyl-2,3-dihydro-benzofuran-2-yl)–imi-
dazole (KU 14R), a close structural efaroxan analogue
able to block its effect, an I3 antagonist [18,20].
However, some recent data have yielded intriguing
results: even high doses of efaroxan did not increase
circulating insulin in mouse [21] and the selective I2
ligand: 2-(2-benzofuranyl)-2-imidazoline (2-BFI) releases
insulin from isolated rat islets [10]. Considering the het-
erogeneity of imidazoline binding sites [22] and that the
putative I3 binding site encompasses a nebulous group of
loci, we have tried to re-evaluate the effect of imidazoline
ligands on insulin release in rabbits. The rabbit was chosen
as a suitable model in view of the paucity of this type of
data for an animal species otherwise very rich in
I2-binding sites [17,23,24]. 2-(4,5-dihydroimidazol-2-yl)-
quinoline (BU 224), considered a selective I2 ligand [25-27],
idazoxan (a typical 2-adrenoceptor antagonist and I2
ligand), methoxy-idazoxan (an 2-adrenoceptor antago-
nist) and efaroxan (I3 putative ligand) were employed in
both in vitro and in vivo experiments to delineate insulin
secretion and glycaemic control.
2.1. Chemicals and Solutions
Forskolin, diazoxide, tolbutamide, methoxy-idazoxan,
nimodipine, yohimbine, 3-isobutyl-1-methylxanthine (IB
MX), chelerythrine and pargyline were provided by Sigma-
Aldrich (Spain); idazoxan was obtained from Rekilt-
Colman Pharmaceutical Company (Germany); brimonidine
(UK 14,304) came from Pfizer (UK); 2-(4,5-dihydroi-
midazol-2-yl)-quinoline (BU 224 hydrochloride), efaroxan
hydrochloride, and 2-(2-ethyl-2,3-dihydro-benzofuran-2-yl)
-imidazole (KU 14R) were obtained from Tocris (Bristol,
UK), calphostin and Rp-Adenosine-3,5-cyclic mono-
phosphothioate triethylamine (Rp-cAMPS) were from
Bionova (Spain). Forskolin and chelerythrine were pre-
pared in DMSO, and final concentrations of DMSO were
0.1% or less in each case.
2.2. Animals
The experiments were performed using male New Zea-
land white rabbits aged 7-12 months (body weight be-
tween 2.5-3.5 kg). The animals were maintained in a 12 h
light-dark cycle and were provided with free access to
food and water. The study was conducted in accordance
with the European Communities Council Directives for
experimental animal care.
2.3. In Vitro Experiments
2.3.1. Islet Isolation and Incubation
The rabbits were sacrificed after the induction of general
anaesthesia with 30 mg kg-1 i.v. of sodium pentobarbital
(Abbott, Spain). The pancreas was removed and disten-
ded with bicarbonate-buffered physiological salt solution.
The islets were isolated by collagenase (Inmunogenetic,
Spain) and hand-picked using a glass loop pipette under a
stereo microscope. They were free of visible exocrine
contamination. The medium used for islet isolation was a
bicarbonate-buffered solution containing 120 mM NaCl,
4.8 mM KCl, 2.5 mM CaCl2, 1.2 mM MgCl2, 5 mM
HEPES and 24 mM NaHCO3. It was gassed with O2-CO2
(94: 6) to maintain a pH of 7.4 and was supplemented with
1 mg ml-1 BSA and 10 mM glucose. When the concentra-
tion of KCl was increased to 30 mM, that of NaCl was
decreased accordingly. The concentration of glucose was
adjusted and test substances were added as required.
2.3.2. Measurements of Insulin Secretion
After isolation, in the first type of experiments, the islets
were pre-incubated for 60 min in a medium containing 15
mM glucose before being distributed into batches of three.
Each batch of islets was then incubated for 60 min in 1 ml
at 37º C of medium containing 8 mM glucose and test
substances, Pargyline was added to the preincubation
medium 40 min before incubation. A portion of the medi-
um was withdrawn at the end of the incubation and its
insulin content was measured by a double antibody-RIA
(insulin CT, Schering, Spain).
In the other type of experiment, the isolated islets were
divided in equal batches of 45-50 and placed in a parallel
perifusion chamber at 37º C and perifused for 30 min
before the start of the experiment at a flow rate of 1.1 ml
min-1. After a 30 min stabilisation period they were
perifused with 8 mM glucose and the appropriate com-
pounds as indicated in the figure legends. Effluent frac-
tions collected at 2 min intervals were chilled until their
insulin content was measured by RIA.
2.2.3. In Vivo Experiments
The experimental design carried out on conscious 24 h
fasted animals has been fully described in other publica-
tions [28, 29]. Arterial blood was sampled by means of an
indwelling cannula placed in the central artery of one ear.
Two control samples, separated by an interval of 30 min,
were taken before drug infusion started. Drug solutions
were infused at a constant rate (0.15 ml min-1) for 30 min
through an indwelling cannula, which was kept functional
by a slow constant infusion of physiological saline (0.07
ml min-1). Plasma glucose was estimated by means of the
glucose oxidase procedure using a kit from Atom (Madrid,
Spain). Insulin was determined by using a radioimmu-
noassay kit (Schering, Spain), with human insulin as
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3.2. Effects of Imidazoline Ligands and
2-Adrenoceptor Antagonists on Insulin
Release from Isolated Islets. Glucose
2.2.4. Statistics
Statistical significance was determined using the Stu-
dent’s t test for unpaired data or analysis of variance in
conjunction with the Newman-Keuls test for unpaired
data. A P value 0.05 was taken as significant. Values
presented in the Figures and Results represent means ±
s.e.m. of at least 6 observations.
When islets were incubated in 8 mM glucose, meth-
oxy-idazoxan in the range 10 µM to 1 mM was unable to
evoke insulin release. However, idazoxan at the same
concentration range induced a clear dose dependent in-
crease in insulin secretion (Figure 2A). Similar results
were found when islets were incubated in the presence of
efaroxan and BU 224 (1-100 µM, Figure 2B). As a
marked significant increase in insulin release was observed
3.1. Effects of Imidazoline Ligands on
Insulin Release in Perifused Islets
The time course of the effects of imidazoline ligands on at 100 µM of either ligand, this particular imidazoline
drug concentration was used for further studies.
insulin release was studied in perifused islets. Idazoxan,
BU 224 and efaroxan, each at the equivalent dose of 100
µM, potentiated the insulin secretory response induced by
8 mM glucose (Figure 1).
Interestingly, the inhibitory effect on glucose induced
insulin release mediated by 1 µM of the selective 2-adre-
noceptor agonist brimonidine (BRM, 55% reduction) was
Time (min)
Gluc8 mMGluc8 mM
30 36 4248 5460 66 7278 8490
Insulin Secretion (pIU per islet min
32 3640 44 4852 5660 64 68 72 768030
Time (min)
Gluc8 mM
-- BU 224 100µM
Gluc8 mM
InsulinSecretion (pIU perisletmin
-- IDZ 100µM --EFX 100µM
Gluc8 mMGluc8 mM
Figure 1. Effects of imidazolines on insulin release from rabbit perifused islets.
Groups of 40 islets were perifused throughout the experiment with a medium
containing 8 mM glucose (-X-). Test substances were introduced between 40
and 70min: (A) 100 µM idazoxan (--) or 100 µM efaroxan (--); (B) 100
µM BU224 (--). Values are mean±s.e.m. for four to six experiments.
Openly accessible at
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G8mM10µM100µM1 mM
InsulinRelease(% basal)
InsulinRelease(% basal)
BU 224
G8mM 1µM10µM100µM
Figure 2. Dose-dependent effects of idazoxan (IDZ), methoxy-idazoxan (Metho-
xyidz) in (A); efaroxan (EFX) and BU 224 in (B) on insulin release from rabbit
islets incubated in the presence of 8 mM glucose in static condition. Each value
represents the mean±s.e.m. from at least 10 observations. *P<0.05, **P<0.01,
and ***P<0.001 indicate statistically significant percentage increase relative
to 8 mM glucose. Basal insulin release at this particular nutrient concen tration
was: 9.65±1.1 µIU ml-1 islet-1 h-1.
completely reversed by 1 µM idazoxan, thus demonstrat-
ing the dual nature (I2 ligand and 2-adrenoceptor antago-
nist) of this compound. However, the 2-adrenoceptor
agonist clearly blunted the response to BU 224 (Figure 3).
As expected, neither yohimbine nor methoxy-idazoxan
affected the response to glucose, though the latter an-
tagonist blocked the inhibitory response to brimonidine.
It is known that efaroxan is able to potentiate glucose
induced insulin release over the range of 4-10 mM glu-
cose [30]. Therefore, the effects of idazoxan and BU 224
(100 µM) were also investigated at different glucose
concentrations. Both ligands, mimicking efaroxan, en-
hanced insulin secretion from 3-15 mM glucose (Figure
4). In the absence of nutrient these imidazolines failed to
release insulin and they did not modify the maximal
secretory response to 30 mM glucose.
3.3. Interaction with KATP Channels
The effects of the three ligands on glucose induced insulin
secretion were tested in the presence of 250 µM diazoxide.
As expected, the KATP channel opener inhibited the re-
sponse to glucose (a 42.5% reduction) and completely
suppressed the effect of idazoxan. However, both efar-
oxan and BU 224 significantly reversed the inhibitory
effect mediated by the channel agonist (Figure 5A).
Since imidazoline compounds and sulphonylureas
block the KATP channel, though interacting with different
and specific binding sites, the effect of the simultaneous
addition of BU 224 plus tolbutamide on glucose mediated
insulin secretion was also studied. When added alone BU
224 or 200 µM of the sulphonylurea compound both drug
induced significant increases in insulin release (80% and
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Insulin Release (% basal)
Insulin Release (% basal)
Figure 3. Inhibition of glucose induced insulin release by the
α2-adrenoceptor agonist brimonidine (BRM, 1 µM) when added
alone () or in the presence of 100 µM of either idazoxan (, A)
or BU 224 (, B). The effects of both imidazoline ligands (100
µM), the α2-adrenoceptor antagonist yohimbine (YOH, 5 µM
), and methoxy-idazoxan (Methoxyidz, 5 µM) by themselves
are also shown. *P<0.05 and ***P<0.001 represent significant
percentage decrease or increase relative to 8 mM glucose.
++P<0.01 when comparing to BRM alone. Basal insulin release
from rabbit islets in static incubation was 12.32±1.4 µIU ml-1
islet-1 h-1. Data are from at least 12 experiments.
and 51%, respectively). When added together, the insulin
secretory response was clearly enhanced (=190%), thus
the effect found with this drug association was signifi-
cantly higher than the response induced by either drug
alone (data not shown).
Interestingly in fresh isolated rabbit islets, idazoxan did
not block the response to efaroxan. However neither
synergism nor antagonism was found when two imida-
zoline ligands (either efaroxan-idazoxan, efaroxan-BU
224) were added together (Figure 5B).
3.4. Role of Ca2+ on Insulin Release
Induced by Imidazoline Ligands
The calcium channel blocker nimodipine (5 µM) at-
tenuated the insulin secretory response to 8 mM glucose
(by 36.5%, P<0.05) and significantly reduced the
Insulin Release (µIU/islet.ml
+BU 224
G 0mMG 3mMG 8mMG 15mMG 30mM
Figure 4. The effect of idazoxan or BU 224 (100 µM, each)
on insulin release in isolated rabbit pancreatic islets in the
presence of different glucose (G) concentrations. Results are
mean±s.e.m. from at least 14 experiments. **P<0.01 and
***P<0.001, values significantly different relative to their
corresponding glucose concentration.
stimulatory effect of BU 224. Interestingly, nimodipine
abolished the response to idazoxan (inhibitory degree:
67.6%, Figure 6A).
The effects of both ligands were also explored in a
low calcium medium (1 mM Ca2+), but in the presence
of a higher glucose concentration (15 mM). Ca2+ re-
duction significantly attenuated the responses to glucose
and BU 224 (from 11.90±2.10 to 5.35±0.35 µIU ml-1 and
from 23.25±4.30 to 12.45±0.65 µIU ml-1, respectively,
P<0.01), though, interestingly, the per-centage increase
in insulin secretion mediated by the ligand was of
similar magnitude in both media (Figure 6A). Idazoxan
elicited a tiny effect.
3.5. Role of the KATP-Independent Pathway on
Insulin Release Mediated by Imidazolines
The experimental approach followed was as has been
described [31]. The response to 8 mM glucose was al-
ready enhanced when islets were incubated in a medium
containing 30 mM potassium and 250 µM diazoxide
(64.7% increase). BU 224, but neither idazoxan or efar-
oxan, still induced a significant insulin secretory effect
(=80%, P<0.05, Figure 6B).
3.6. Intracellular Sites Involved in the Insulin
Secretory Activity of Imidazolines
Since true I2 ligands are reported to be linked to MAO
enzymes, it was necessary to test the effect of a non-
selective MAO inhibitor on insulin release in the presence
of the three ligands. Pargyline (10 µM) did not modify the
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response to either glucose or any of the ligands under
investigation (Figure 9B).
When either BU 224 or efaroxan was assayed in the
presence of forskolin (10 µM), drug interaction led to an
enhanced insulin secretory response (percentage in-
creases with BU 224, forskolin and both together were:
135.5, 149.7 and 438.2, respectively; similarly, in the case
of efaroxan, the results were: 100.9, 149.7, and 407.9). In
the same way, the stimulatory response to BU 224 was
potentiated by 100 µM of the phosphodiesterase inhibitor
3-isobutyl-1-methylxanthine (IBMX, 312%). Again, the
increase derived from drug combination (association with
either forskolin or IBMX) was significantly higher than
the effect found with any drug alone (Figure 7, Δ to
IBMX alone=177.3±24.5 %).
Neither glucose nor efaroxan and idazoxan induced
insulin release were affected by the presence of Rp-
Insulin Release (% Basal)
Insulin Release (% Basal)
Figure 5. (A): Reversal of diazoxide induced inhibition of
secretion by imidazoline ligands. 250 µM of the channel opener
were added to islets incubated in 8 mM glucose in the absence
() or presence () of 100µM of the three different ligands:
idazoxan, BU 224 and efaroxan. *P<0.05 statistically signifi-
cant percentage reduction relative to glucose; +++P<0.001
when comparing to diazoxide. Basal insulin release was 6.7±0.8
µIU ml-1 islet-1 h-1. Each value represents the mean±s.e.m. from
at least 12 experiments. (B): Insulin release from isolated islets
in the presence of the different imidazoline ligands added either
separately () or together () as shown. 100 µM of each ligand
was always applied. Basal insulin release in the presence of 8
mM glucose was: 7.45±0.55 µIU ml-1 islet-1 h-1. Data represent
the mean from at least 17 observations.
Insulin Release (% Basal)
G 8mMNimoBU 224 BU+NimoIDZIDZ+NimoG 15mMBU 224IDZ
Insulin Release (% Basal)
Insulin Release (% basal)
With K
Figure 6. (A): Effects of BU 224 and idazoxan on insulin release
in the absence () and presence () of 5 µM nimodipine
*P<0.05 represents statistically significant percentage inhibition
relative to glucose; +P<0.05, percentage when comparing to
either ligand. Basal insulin release was: 7.6±0.6 µIU ml-1 islet-1
h-1. On the right: rabbit islets were incubated in a low calcium
medium (1 mM) but with a higher glucose concentration (15
mM). In these experimental conditions insulin release was
5.35±0.3 µIU ml-1 islet-1 h-1. The effects of BU 224 and IDZ are
shown. (B): The insulin secretory responses induced by BU 224,
idazoxan and efaroxan in the presence of 30 mM KCl and 250
µM diazoxide (). The effect found in normal medium () is
also presented for comparison. *P<0.05, significant percentage
increase relative to normal medium; +P<0.05, percentage in-
crease when compare to insulin release from the medium en-
riched whit KCl and diazoxide. Basal insulin secretion in normal
medium: 9.1±0.9 µIU ml-1 islet-1 h
-1. Data from these different
experimental designs come from at least 14 observations.
Adenosine-3,5-cyclic monophosphothioate triethylamine
(Rp-cAMPS, 200 µM). This PKA inhibitor did attenuate
the effect of BU 224, though a residual significant in-
crease of 34% above basal levels was still observed with
this ligand (Figure 8A). The PKC inhibitor chelerytrine
1µM [32] did not alter glucose induced insulin release
though significantly blocked the response to BU 224
Figure 8B). However insulin secretion in the presence of
idazoxan or efaroxan was not modified by chelerytrine.
Similarly, calphostin (1 µM), another Protein-Kinase C
inhibitor, completely suppressed the effect of (BU 224
(from 21.32.9 to 11.91.7 µIU ml-1 islet–1 h
-1, insulin
secretion in the presence of 8 mM glucose being=
11.61.4 µIU ml-1 islet –1 h-1), the response to efaroxan
(22.24.7 and 22.04.4 µIU ml-1 islet–1 h
-1 remaining
unaltered in the absence and presence of the inhibitor.
Data are from at least 12 experiments).
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Insulin Release (% Basal)
*** ***
** **
Figure 7. Stimulation of insulin release by BU 224 and efaroxan
when added alone () or in the presence of either 10 µM forsko-
lin or 100 µM 3-isobutyl-1-methylxanthine (IBMX, ). Rabbit
islets were incubated in 8 mM glucose throughout the experi-
ment. **P<0.01 and ***P<0.001 represent significant percent-
age increase relative to glucose; ++P<0.01 and +++P<0.001,
synergistic percentage increase. Basal insulin release: 7.1±1.0
µIU ml-1 islet-1 h-1. Data are from at least 14 observations.
Figure 8. (A): Stimulatory effect on insulin secretion induced by
idazoxan, BU 224 and efaroxan when added alone () (100 µM,
of either ligand) or in the presence of 200 µM of the selective
PKA inhibitor Rp-cAMPS (). (B): The insulin secretory re-
sponse of the three imidazoline ligands, alone () or in the pres-
ence of 1 µM of the PKC inhibitor Chelerythrine (). The effects
of both inhibitors on glucose induced insulin release are also
shown. *P<0.05 and **P<0.01, significant percentage increase
relative to glucose; +P<0.05 and ++P<0.01 when comparing to
ligand alone. Basal insulin release: (A) 12.9 ±1.3 and (B) 11.9
±1.1 µIU ml-1 islet-1 h-1. Data are from at least 13 observations.
The compound 2-(2-ethyl-2,3-dihydro-benzofuran-2-
yl)-imidazole (KU 14R, 100 µM) did not alter glucose
induced insulin release, but selectively reduced the effect
of BU 224 (from a 124.5% to a 30.5% increase), therefore,
responses to idazoxan and efaroxan remained unchanged.
This reported antagonist also lowered forskolin induced
insulin secretion (from a 329% to a 158% increase,
P<0.05), (Figure 9A).
Insulin Release (% Basal)
Insulin Release (% Basal)
+ Pargilyne
+KU 14R
Figure 9. (A): The effect of the compound KU 14R (100 µM)
on insulin release induced by the three imidazoline ligands and
10 µM forskolin. (B): The insulin secretory effect of three
imidazoline ligands in the absence () and presence () of the
dual MAO inhibitor pargyline (10 µM). Basal insulin secretion
when islets were bathed in 8 mM glucose: (A) 7.8±0.8 and (B)
5.6±0.5 µIU ml-1 islet-1 h-1. *P<0.05 and **P<0.01, significant
percentage increase relative to glucose. +P<0.05 and +++P
<0.001, when compared to secretagogue alone. Data were ob-
tained from at least 17 observations.
3.7. Effects in the Presence of Yohimbine,
Idazoxan, BU 224 and Efaroxan on
Intravenous Glucose Tolerance Test
and Plasma Insulin Levels, Studies
in Conscious Fasted Rabbits
When infused alone at the equivalent dose of 10 µg kg-1
min-1, neither yohimbine [29], nor idazoxan modified
basal values of either plasma glucose or circulating insu-
lin (Figure 10). Interestingly, after the administration of
BU 224 at the same dose, the ligand induced a progres-
sive, persistent and significant increase in plasma insulin
(at 45 min=184.40±42.95%, n=5, P<0.05, vs.
1.27±6.75%, n=7 in saline treated animals, see Figure
11). The pre-infusion plasma glucose level was:
6.25±0.80 mM, n=5. These levels were not significantly
modified after drug infusion.
Pre-treatment with yohimbine did not modify the re-
sponses to a glucose load (10 mg kg-1 min-1), (Figure 10).
in plasma glucose at 30 min in the absence and pres-
ence of yohimbine was, respectively, 3.87±0.3 mM, n=8,
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ΔPlasma Glucose (mM)
-30015 3045 6090
ΔIRI (% basal)
-30015 30 45 6090
Time (min)
*** **
5.0 C
-3001530 456090
-3001530 45 6090
Time (min)
*** **
Figure 10. Effects of a glucose load (10 mg kg-1 min-1) on plasma glucose (A and C) and
circulating insulin levels (B and D) in the absence (--) and presence of yohimbine (left)
or idazoxan (right) (--) in conscious fasted rabbits. The effects of saline (-X-), yohim-
bine and idazoxan (--, 10 µg kg-1 min-1) by themselves on both parameters are also pre-
sented; saline or drugs were administered for 30min (white horizontal bar) alone or before
a 30min i.v. glucose load (black bar). Ordinate scales, mM plasma glucose refers to the
variations from control values. IRI levels are expressed as percentage changes from the
control level (control=100%). Each point of any given curve represents the mean±s.e.m. for
at least 6 rabbits. Vertical lines indicate s.e.m. **P<0.01 and ***P<0.001, values significant-
ly different between glucose and saline. +P<0.05 and +++P< 0.001, values significantly diff-
erent between glucose vs. glucose+idazoxan.
vs. 4.17±0.75 mM, n=6, N.S. in immunoreactive insu-
lin (IRI) plasma levels, also at 30 min, in the absence and
presence of the drug were: 226.55±40.55%, n=8, vs.
380±102%, n=6. No significant difference in the area
under the insulin curve was found between glucose and
yohimbine pre-treated animals (98.5±27.93 vs. 141.06±
31.22 µIU ml-1 h-1, n=6).
However, in the presence of idazoxan (10 µg kg-1
min-1), glucose evoked a greater rise in IRI levels, (Fig-
ure 10) (at 30 min=1032.90±278.60%, n=6, P<0.01
when compared to glucose alone). A significant reduction
in plasma glucose was also observed (at 15 and 30 min
in the presence of the drug being 1.22±0.38 and 2.98±0.4
mM, n=6, P<0.05).
When the glucose load was assayed in animals
pre-treated with BU 224, this ligand induced both a re-
duction in plasma glucose (at 30 min in the absence and
presence of the drug being: 3.40±0.35 mM, n=6 vs.
2.30±0.45, n=6, P<0.05) and a greater increase in IRI
plasma levels, (Figure 11) (similarly, at 30 min was:
116.60±28.10%, n=6 vs. 325.65±59.05%, n=8, P<0.001).
Finally, efaroxan alone induced an increase in circulat-
ing insulin (at 30 min=196.9%, mean of 2 animals vs.
-2.90±2.85%, n=7, in saline control rabbits) which per-
sisted for 90 min (at 60 min=603.65±260%). When
administered before glucose it also enhanced insulin se-
cretion (at 30 min=356.35±60%, n=2 vs. 116.60± 28.10,
n=6 in glucose treated animals). However, this insulin
secretory effect persisted for as long as 9 h, the animals
remaining in hypoglycaemia. Therefore, in vivo experi-
ments with this molecule were discontinued.
Pre-infusion absolute values of arterial plasma glucose
and circulating insulin for saline, glucose and drug treated
animals ranged between 4.56±05 and 6.62±0.25 mM, and
5.65±1.60 and 12.15±2.70 µIU ml-1, respectively.
M. J. García-Barrado et al. / HEALTH 2 (2010) 112-123
SciRes Copyright © 2010 Openly accessible at http://www.scirp.org/journal/HEALTH/
-1. 0
-30015 30 456090
plasma glucose(mM)
BU 224
-30015 30 456090
IRI (% basal)
Time (min)
Figure 11. Changes in plasma glucose (A) and in immunoreactive
insulin (IRI) (B) levels in conscious fasted rabbits, measured
after the i.v. infusion of physiological saline (-X-), glucose alone
(--, 10 mg kg-1min-1), BU 224 (-- 10 µg kg-1min-1) and BU
224+glucose (--); the BU 224 was infused for 30 min (open
bar) just before a 30min glucose infusion (black horizontal bar).
Ordinate scales, mM plasma glucose refers to the variations
from control values. IRI levels are expressed as percentage
changes from the control level (control=100%). Each point of
any given curve represents the mean±s.e.m. for at least 7 rabbits.
*P<0.05 and ***P<0.001, values significantly different be-
tween glucose or BU 224 vs. saline. +P<0.05, ++P<0.01, and
+++P<0.001, values significantly different between glucose vs.
glucose+BU 224.
Studies on insulin secretion have mainly been carried out
using mouse and/or rat isolated islets. However, there is a
lack of experimental data for other animal species, rabbit
included. Since rabbit tissues are very rich in imidazoline
binding sites [17,24] this animal was chosen for the present
work. The results reported in this study assess the validity
of our model: insulin release from isolated islets was glu-
cose-dependent and very sensitive to changes in the ex-
tracellular concentrations of Ca2+ and K+ ions. Similarly,
conventional stimulatory and/or inhibitory responses (i.e.,
to forskolin, diazoxide) were also confirmed.
Idazoxan induced a very clear dose-dependent insulin
secretory response. These are rather paradoxical results,
since it has been reported that idazoxan is unable to re-
lease insulin in the rat, or it has a weak concentration
independent effect in mouse [13,14]. An even more
I2-selective ligand, BU 224 [27], also evoked a dose-
dependent secretory response, as did the well known and
studied ligand efaroxan [1]. In this model methoxy-idazoxan
assayed over a wide range failed to elicit insulin release,
thus corroborating its nature as a true 2-adrenoceptor
antagonist. This property was also shared by idazoxan and
it was unmasked when tested against the 2-adrenoceptor
agonist brimonidine (UK 14,304). In this way, idazoxan
reflected its established dual behaviour. However, no inter-
action with 2-adrenoceptors could be found with BU 224.
The mode of action of imidazolines is complex. Clas-
sical insulinotropic compounds inhibit KATP channels in
the ß-cell, but, in addition, they exert a direct effect on
exocytosis [7,32]. The three ligands studied behaved as
KATP channel blockers. Interaction of efaroxan with KATP
channels could be inferred from perifusion studies and
when incubating the islets in the presence of diazoxide,
since efaroxan alleviated the suppressive effect on insulin
release induced by the potassium channel opener. Similar
results have been reported for rat islets [30] using other
imidazoline drugs (RX871024, S-22068 [4, 34] and in the
present work with the selective I2-ligand BU 224. How-
ever, diazoxide abolished the response to idazoxan, but
not to BU 224. It is necessary to consider at this time that
in mouse islets idazoxan induces a partial inhibition of
KATP channels, sufficient to depolarize the plasma mem-
brane and to open voltage-dependent Ca2+ channels with
a subsequent modest increase in intracellular calcium
[13]. In the presence of partial channel inhibition, dia-
zoxide, by reducing the binding affinity of the ligand,
could easily suppress the response. It is also noteworthy
that nimodipine similarly abolished the effect of idazoxan
and that just simple reduction in the Ca2+ concentration in
the extracellular medium severely attenuated the ligand
response. An α-1 partial agonist effect of idazoxan has
been recently reported [35]. However there is no evidence
for a α-1 adrenoceptor involvement in insulin secretion in
isolated islets [36]. When rabbit islets were incubated in
the presence of the selective α-1 adrenoceptor agonist,
amidephrine, no insulin secretory response was found
(glucose 8 mM 11.61.4 vs amidefrine 10.52.2 µIU ml-1
islet–1 h-1). In the same experimental situations, BU 224
was still able to increase insulin release.
It is also accepted that this kind of imidazoline com-
pounds block the KAT P channel at the level of the Kir6.2
pore [9]. Our experimental results, studying drug interac-
tions among themselves and in the presence of tolbutamide,
trend to support this notion. Simultaneous addition of
efaroxan and idazoxan, or efaroxan and BU 224, did not
lead to synergism or antagonism, probably considering that
the three ligands shared a common drug binding site at the
pore level [37]. On the other hand, an additive effect was
found when BU 224 and tolbutamide were administered
together. Indeed, the imidazoline could not displace the
M. J. García-Barrado et al. / HEALTH 2 (2010) 112-123
SciRes Copyright © 2010 Openly accessible at http://www.scirp.org/journal/HEALTH/
sulphonylurea from its SUR1 receptor. The ensuing en-
hanced response could result from additive effects at the
KATP channel, or by BU 224 activation of a signal-
transduction pathway (see below). Similar results have
been reported with other imidazolines [14,38].
It is also well established that insulin secretion in the
presence of these compounds exhibited glucose depend-
ency [8,38]. Identical results, expressing the requirement
for a high energy state of the cell (high ATP/ADP ratio)
have been found with the I2-ligands used in this work.
Studies with permeabilised islets and HIT T15 cells have
revealed a direct effect of imidazolines (RX871024,
BL11282) on exocytosis independent of KAT P channel
activity. PKA and PKC, with subsequent activation of
protein phosphorylation/dephosphorylation steps, would
play a central role in the regulation of this process [8].
However, these kinase inhibitors failed to alter the re-
sponse to efaroxan and idazoxan [12]. Our results, using
either PKA/PKC inhibitors or excess K+, confirm that
efaroxan and idazoxan induced insulin secretion is de-
pendent of KAT P channel activity, whereas the effect of BU
224 requires, in addition, PK activation. A synergism be-
tween the effect of BU 224 and either forskolin or IBMX
on insulin secretion was also evident, suggesting the per-
missive role of PKA activity on this particular response.
Interestingly, the compound KU 14R, known as an
efaroxan antagonist [39], did not alter, in the present work,
the response to this ligand, though it blocked the effect of
BU 224, significantly attenuating the response to forsko-
lin [40]. A lack of antagonism between efaroxan and KU
14R has also been reported recently in mouse islets [41].
Consequently an association among BU 224-PKA-KU
14R could be inferred in our model. It is noteworthy that
at the concentrations used in the present work BU 224
behaved as a reversible inhibitor of MAO A and B, pre-
venting hydrogen peroxidase production in adipose tissue
[42]. However, in our model MAO inhibition did not
modify glucose or BU 224 mediated insulin release. At
this point it is interesting to note that the total capacity of
the pancreas to oxidise MAO substrates was limited
compared with the overall mass and amine oxidase ac-
tivities of muscular and adipose tissue [43]. Therefore
these results reassess the true nature of BU 224 as an I2
ligand, though the response under study seems to be
independent of MAO binding sites. It has been reported
recently that selective I2-ligands can bind creatine kinase
[44,45], a key enzyme important for ATP synthesis. This
additional interaction would help to understand the
mechanism(s) of BU 224 induced insulin release, con-
sidering the importance of ATP for exocytosis even at
stages distal to an increase in [Ca+2]i (see experiments at
high concentrations of K+).
The presence of I2 binding sites (IBS) mediating the ef-
fects of these ligands has not been accepted on the basis of
the failure of idazoxan to elicit insulin secretion, lack of
data with more selective I2-ligands and binding studies
with methoxy-idazoxan. Results presented in this work
refute these premises. In addition the ligand BTS 67582
can bind to the I2 imidazoline receptor with potency con-
sistent with its effect on insulin secretion [46]. The drug
could regulate insulin release by an interaction with the
KATP channel or by exerting a direct effect in the process of
exocytosis [46,47]. Curiously indeed, idazoxan and BU
224 also increase insulin release, blocking KATP channel
activity at a site shared by the third imidazoline ligand
efaroxan. Therefore, considering that a number of
I2-ligands can bind a common site on the channel, this
binding site, independent of MAO activity, might be con-
sidered as a variant or subtype of the classic I2-binding site.
It is also known that the presence of I2 sites on MAO en-
zyme can not satisfactorily represent the diverse biological
targets of I2-ligands [48,49]. Intracellular binding sites
linked to protein kinase(s) activation (I3-receptor?) would
also be involved in the amplifying effect of BU 224.
In vivo studies reassess in vitro data. Idazoxan and BU
224, but not yohimbine, enhanced the insulin secretory
response to a glucose load. Temporal patterns of insulin
secretion when BU 224 was infused alone or in the pres-
ence of glucose showed the complex behaviour of this
molecule: its glucose dependency as well as its interaction
with KATP dependent and independent mechanisms. When
comparing circulating levels of insulin after a glucose
challenge in animals pretreated with any of the three drugs,
idazoxan was able to induce the maximal response. The
dual nature of this ligand should be borne in mind: its
ability to block: 1) pre and post-synaptic 2 adrenoceptors
and thus increase plasma catecholamine levels [50], with a
subsequent -adrenoceptor mediated effect; and 2) KATP
channels as an I-ligand (like BU 224). Combined mecha-
nisms would be responsible for such an effect.
Though BU 224 showed a greater antihyperglycaemic
effect than idazoxan, the effect was lower than expected
considering its ability to release insulin and to restrain
lipolysis [42]. However this molecule, being an MAO
inhibitor, should prevent metabolic inactivation of en-
dogenous catecholamines, thus enhancing intrinsic
sympathomimetic activity. Non-selective blocking of
Kir6.2 affecting channels at several locations could also
unmask compensatory responses able to attenuate the
blood glucose lowering response.
The imidazoline ligands interacting with either I2 or
I3-binding sites would mediate in vitro, as well as in vivo,
insulin secretion. However, additional extrapancreatic
sites of action would attenuate their antihyperglycaemic
effect. In conclusion, the administration of BU 244 in-
M. J. García-Barrado et al. / HEALTH 2 (2010) 112-123
SciRes Copyright © 2010 Openly accessible at http://www.scirp.org/journal/HEALTH/
duces extended insulin release that would produce po-
tential hypoglycaemia. This could be an adverse effect
whether this molecule is used as antidiabetic drug.
The authors express gratitude to Mr. G.H. Jenkins for his help with the
English version of the manuscript. We are very grateful for the fi-
nancial support from Junta de Castilla y León (JCYL), grant nº
SA42/00B (Spain).
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