Title page Effects of the Combination of a Dipeptidyl...
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Title page
Effects of the Combination of a Dipeptidyl Peptidase IV Inhibitor and an Insulin
Secretagogue on Glucose and Insulin Levels in Mice and Rats
Kazuto Yamazaki, Nobuyuki Yasuda, Takashi Inoue, Eiichi Yamamoto, Yukiko Sugaya,
Tadashi Nagakura, Masanobu Shinoda, Richard Clark, Takao Saeki and Isao Tanaka
Tsukuba Research Laboratories, Eisai Co., Ltd., 5-1-3, Tokodai, Tsukuba, Ibaraki
300-2635, Japan
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Copyright 2006 by the American Society for Pharmacology and Experimental Therapeutics.
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Running title page
Running title: Combination of DPP-IV inhibitor and insulin secretagogue
Corresponding author: Kazuto Yamazaki; Tsukuba Research Laboratories, Eisai Co.,
Ltd., 5-1-3, Tokodai, Tsukuba, Ibaraki 300-2635, Japan; Tel: +81-29-847-5817, Fax:
+81-29-847-5367; E-mail: [email protected]
Number of text pages: 23
Number of tables: 0
Numbers of figures: 8
Numbers of references: 31
Number of words in the Abstract: 208
Number of words in the Introduction: 405
Number of words in the Discussion: 836
Abbreviations: ANOVA, analysis of variance; AUC, area under the curve; DPP-IV,
dipeptidyl peptidase IV; E3024,
3-but-2-ynyl-5-methyl-2-piperazin-1-yl-3,5-dihydro-4H- imidazo[4,5-d]pyridazin-4-one
tosylate; GIP, glucose-dependent insulinotropic polypeptide; GLP-1, glucagon-like
peptide-1; GPCR, guanine nucleotide-binding protein-coupled receptor; OGTT, oral
glucose tolerance test; P32/98,
di-[(2S,3S)-2-amino-3-methyl-pentanoic-1,3-thiazolidide] fumarate; SUR1,
sulfonylurea receptor type 1; KATP channel, ATP-sensitive potassium channel; PKA,
protein kinase A; cAMP-GEFII, cAMP-regulated guanine nucleotide exchange factor
II; Rim2, Rab3-interacting molecule 2 (GIP)
Recommended section: Endocrine and Diabetes
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Abstract
Several combination therapies have been tried for treating of type 2 diabetes in order to
control more effectively fasting hyperglycemia and postprandial hyperglycemia. In this
study, we have examined the effects of combinating a novel, selective and competitive
dipeptidyl peptidase IV (DPP-IV) inhibitor, E3024
(3-but-2-ynyl-5-methyl-2-piperazin-1-yl-3,5-dihydro-4H-imidazo[4,5-d]pyridazin-4-on
e tosylate) with a representative of one of two types of insulin secretagogues, i.e. either
glybenclamide (a sulfonylurea) or nateglinide (a rapid-onset/short-duration insulin
secretagogue), on glucose and insulin levels in an oral glucose tolerance test (OGTT)
using mice fed a high-fat diet. In addition, we have investigated the effects of these
combinations on blood glucose levels in fasting rats. Two-way analysis of variance
showed that the combination of E3024 and glybenclamide improved glucose tolerance
additively, and also caused a synergistic increase in insulin levels in the OGTT in mice
fed a high-fat diet. In a similar way, the combination of E3024 and nateglinide
ameliorated glucose tolerance additively and raised insulin levels additively. In fasting
rats, co-administration of E3024 with glybenclamide or nateglinide treatment did not
affect the glucose-lowering effects of the insulin secretagogues. Therefore, a DPP-IV
inhibitor in combination with glybenclamide or nateglinide may be a promising option
for the treatment of type 2 diabetes, and particularly for controlling postprandial
hyperglycemia in the clinic.
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Introduction
Dipeptidyl peptidase IV (DPP-IV) degrades active glucagon-like peptide-1
(GLP-1 (7-36)amide and GLP-1 (7-37)), which is an incretin released from L cells in
the intestine after meal intake that promotes insulin secretion in a glucose-dependent
manner. GLP-1 has an antidiabetic action in patients with type 2 diabetes (Gutniak et al.,
1994; Nauck et al., 1993). DPP-IV cleaves GLP-1 rapidly so the latter’s half-life is only
1-2 min. Accordingly, the prevention of GLP-1 inactivation by DPP-IV inhibition is
currently being actively explored as a novel approach to the treatment of type 2 diabetes
(Deacon, et al. 2004). DPP-IV inhibition leads to blood glucose-lowering effects in
animal models of diabetes (Perderson et al., 1998; Reimer et al., 2002; Burkey et al.,
2005), and in patients with type 2 diabetes (Ahren et al., 2002; 2005).
E3024
(3-but-2-ynyl-5-methyl-2-piperazin-1-yl-3,5-dihydro-4H-imidazo[4,5-d]pyridazin-4-on
e tosylate) is a novel, selective and competitive DPP-IV inhibitor, discovered in our
laboratories (Yasuda et al., 2006). Demuth et al. (2005) have classified DPP-IV
inhibitors based on their mode of inhibition and structures as follows: reversible product
analog inhibitors (e.g. P32/98 (Sorbera, et al., 2001)), covalently modifying product
analog inhibitors (e.g. vildagliptin (LAF237) (Villhauer et al., 2003)), and reversible
non-peptidic heterocyclic inhibitors (e.g. sitagliptin (MK-0431) (Kim et al., 2005)).
E3024 belongs to the third group, and has a novel, imidazopyridazinone structure.
Insulin secretagogues used in the clinic are categorized into the following two
groups: sulfonylureas and rapid-onset/short-duration insulinotropic agents (Krentz and
Bailey, 2005). The former class includes glybenclamide and glyburide, while the latter
includes nateglinide and repaglinide. The sulfonylureas target fasting hyperglycemia,
while the rapid-acting insulin secretagogues improve postprandial hyperglycemia (Van
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Gaal and De Leeuw, 2003).
Recently, the importance of reducing not only fasting hyperglycemia, but also
postprandial hyperglycemia, has been demonstrated. Fasting hyperglycemia increases a
risk of microvascular complications (UK Prospective Diabetes Study (UKPDS) Group,
1998), while postprandial hyperglycemia has shown to be an independent risk factor for
the development of macrovascular complications (Monnier, 2000; Ceriello, 2000;
Temelkokova-Kurktschiev, 2000). Several combination therapies employing agents
with complementary mode of actions have been examined to address improvement of
fasting hyperglycemia and postprandial hyperglycemia. In this study, we examined the
effects of a combination of E3024 and glybenclamide, and that of E3024 and
nateglinide on glucose and insulin levels in an oral glucose tolerance test in mice fed a
high-fat diet, and further studied the effects of these combinations on blood glucose
levels in fasting rats to explore their possibilities as novel combination therapies.
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Materials and Methods
Chemicals. E3024 was synthesized in our laboratories. Glybenclamide
(4-[2-(5-chloro-2-methoxybenzoylamino)ethyl](N-cyclohexylcarbamoyl)benzene
sulfonamide) was purchased from Sigma (St. Louis, MO). Nateglinide
((-)-N-(trans-4-isopropylcyclohexanecarbonyl)-D-phenylalanine) was purchased from
Yamanouchi Pharmaceutical Co., Ltd. (Tokyo, Japan). The chemical structure of E3024
is indicated in Figure 1. GLP-1 (7-36)amide and human glucose-dependent
insulinotropic polypeptide (GIP) were purchased from Bachem AG (Bubendorf,
Switzerland) and Peptide Institute, Inc. (Osaka, Japan), respectively.
Animals. Ten-week-old male C57BL/6NCrj mice and five-week-old male Wistar rats
were purchased from Charles River Japan (Tokyo, Japan). The mice and rats were
provided with a commercial diet (MF, Oriental Yeast, Tokyo, Japan) and water ad
libitum and were kept under conventional conditions of controlled temperature,
humidity and lighting (22 ± 2°C, 55 ± 5% and a 12-hr light/dark cycle with lights on at
07:00 a.m.). All procedures were conducted according to the Eisai Animal Care
Committee’s guideline.
Effects of the combination of E3024 and glybenclamide or nateglinide on blood
glucose levels in an oral glucose tolerance test (OGTT). Mice were fed a high-fat diet
(D12492 Rodent Diet with 60 kcal% fat; Research Diets, Inc., NJ) for four weeks from
11 weeks of age, and 28 mice were selected based on body weight and randomly
divided into four groups. The reason why we used the high-fat diet-fed mouse model is
that this model is considered to be a robust model for impaired glucose tolerance and
early type 2 diabetes (Winzell and Ahrén, 2004), both of which are targets of DPP-IV
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inhibitors. E3024 (3 mg/kg), glybenclamide (1 mg/kg), a mixture of E3024 (3 mg/kg)
and glybenclamide (1 mg/kg), or vehicle (0.5% methylcellulose (MC)) alone was orally
administered to overnight-fasted mice 0.5 hr prior to an oral glucose load (2 g/kg).
Blood samples were collected from the tail vein 0.5 hr before the glucose load, and 0,
0.5, 1 and 2 hr after the glucose load, and blood glucose and plasma insulin levels were
determined. The same OGTT procedure was performed for the combination of E3024
and nateglinide, but the dose of nateglinide was 50 mg/kg.
Effects of the combination of glybenclamide and nateglinide on blood glucose
levels in the OGTT. Mice were fed a high-fat diet for five weeks from 11 weeks of age,
and 20 mice were selected based on body weight and randomly divided into four groups.
Glybenclamide (1 mg/kg), nateglinide (50 mg/kg), a mixture of glybenclamide (1
mg/kg) and nateglinide (50 mg/kg), or vehicle (0.5% MC) alone was orally
administered to overnight-fasted mice 0.5 hr prior to the oral glucose load (2 g/kg).
Blood samples were collected from the tail vein 0.5 hr before the glucose load, and 0,
0.5, 1 and 2 hr after the glucose load, and blood glucose levels were determined.
Effects of the combination of glybenclamide and GLP-1 on blood glucose levels in
the OGTT. Mice were fed a high-fat diet for six weeks from 11 weeks of age, and 24
mice were selected based on body weight and randomly divided into four groups.
Before the glucose load (2 g/kg), glybenclamide (1 mg/kg) or vehicle (0.5% MC) alone
was orally administered to overnight-fasted mice. GLP-1 (7-36)amide (1 µg) or saline
was intraperitoneally injected immediately after the glucose administration. Blood
samples were collected from the tail vein 0.5 hr before the glucose load, and 0, 0.5, 1
and 2 hr after the glucose load, and blood glucose levels were determined.
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Effects of the combination of glybenclamide and GIP on blood glucose levels in the
OGTT. Mice were fed a high-fat diet for six weeks from 11 weeks of age, and 20 mice
were selected based on body weight and randomly divided into four groups. Before the
glucose load (2 g/kg), glybenclamide (1 mg/kg) or vehicle (0.5% MC) alone was orally
administered to overnight-fasted mice. GIP (1 µg) or saline was intraperitoneally
injected immediately after the glucose administration. Blood samples were collected
from the tail vein 0.5 hr before the glucose load, and 0, 0.5, 1 and 2 hr after the glucose
load, and blood glucose levels were determined.
Effects of the combination of E3024 and glybenclamide or nateglinide on fasting
blood glucose levels in normal rats. Six-week-old rats were used. E3024 (3 mg/kg),
glybenclamide (1 mg/kg), a mixture of E3024 (3 mg/kg) and glybenclamide (1 mg/kg),
or vehicle (0.5% MC) alone was orally administered to overnight-fasted rats. Blood
samples were collected from the tail vein before (0 hr), and 1, 2 and 3 hr after the drug
administration in order to determine the glucose levels. The same procedures were
performed for the combination of E3024 and nateglinide, but the dose of nateglinide
was 50 mg/kg.
Blood glucose and plasma insulin determination. Blood samples (10 µL) were
collected from the tail vein and mixed with 140 µL of 0.6 mol/L perchloric acid. After
centrifugation (3,000 rpm; 10 min; room temperature), the supernatants were assayed
for glucose using an enzymatic assay kit (Glucose CII-test WAKO, Wako Pure
Chemicals) with a microplate spectrophotometer (SpectraMax; Molecular Devices, CA).
Blood samples (approximately 50 µL) were collected from the tail vein with
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heparinized capillary tubes and centrifugated (7,000 rpm; 5 min; 4°C). The supernatants
were assayed for plasma insulin levels using an enzyme-linked immunosorbent assay kit
(Ultra sensitive rat insulin ELISA kit, Morinaga Institute of Biological Science,
Kanagawa, Japan) and mouse insulin (Morinaga Institute of Biological Science) as a
standard with the microplate spectrophotometer.
Statistical analysis. Data are expressed as the mean ± standard error of the mean
(S.E.M.). To determine the integrated glucose response to the oral glucose challenge,
the area under the curve (AUC) of delta blood glucose after the oral glucose load was
calculated using a trapezoidal rule. To examine main effects and interaction in the
combination studies, two-way analysis of variance (ANOVA) was performed on the
AUC values and plasma insulin levels at 0.5 hr. The Tukey multiple comparison test
was used to examine the difference between the two groups.
In the studies on effects of the combination of E3024 and glybenclamide or
nateglinide on fasting blood glucose levels, we performed two-way ANOVA with
respect to the values of delta blood glucose at 2 hr (that is, the differences in blood
glucose levels between 0 and 2 hr) in the combination with glybenclamide, and those at
1 hr in the combination with nateglinide.
A probability (p) value <0.05 (two-sided) was considered statistically significant.
Statistical analyses were performed using an SAS software package version 8.1 (SAS
Institute Japan Ltd., Tokyo, Japan).
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Results
Figure 2 illustrates the changes of blood glucose levels (A) and the AUC of delta
blood glucose (B) in the OGTT using mice fed the high-fat diet that were treated with
E3024 (3 mg/kg) and/or glybenclamide (1 mg/kg). Two-way ANOVA indicated
significant main effects of both E3024 and glybenclamide on the AUC values. No
significant interaction was seen between the effects of E3024 and glybenclamide (Fig.
2B). E3024 and glybenclamide significantly reduced the AUC values, compared with
that of the vehicle treatment. The AUC value of the combination group was
significantly lower than those of the other groups. Figure 2C shows the changes of
plasma insulin levels in the mice. Two-way ANOVA indicated significant main effects
of E3024 and glybenclamide on plasma insulin levels 0.5 hr after the oral glucose load.
In addition, a significant interaction was detected between the effects of E3024 and
glybenclamide. The insulin level of the E3024 plus glybenclamide treatment was
significantly higher than those of the other three groups.
Figure 3 shows the changes of blood glucose levels (A) and the AUC of delta
blood glucose (B) in the OGTT using mice fed the high-fat diet that were treated with
E3024 (3 mg/kg) and/or nateglinide (50 mg/kg). Two-way ANOVA indicated
significant main effects of both E3024 and nateglinide on the AUC values. No
significant interaction was seen between the effects of E3024 and nateglinide (Fig. 3B).
E3024 and nateglinide significantly decreased the AUC values in comparison with that
of the vehicle treatment. The AUC value of the combination group was significantly
lower than those of the vehicle- and nateglinide-treated groups. Figure 3C. shows the
changes of plasma insulin levels in the mice. Two-way ANOVA indicated significant
main effects of both E3024 and nateglinide on plasma insulin levels 0.5 hr after the oral
glucose load. No significant interaction between the effects of E3024 and nateglinide
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was seen. The combination resulted in levels significantly higher than any other group.
Effects of the combination of glybenclamide and GLP-1 on glucose excursions in
the OGTT are shown in Figure 4A. Two-way ANOVA indicates significant main
effects of both glybenclamide and GLP-1 on the AUC values, but there was no
significant interaction between the effects of glybenclamide and GLP-1 (Fig. 4B). Both
GLP-1 alone and glybenclamide alone caused significant reduction of the AUC,
compared with the control. The AUC values of the GLP-1 plus glybenclamide treatment
were significantly lower than those of treatment with either GLP-1 or glybenclamide
alone. Likewise, we also observed an additive effect of improvement of glucose
tolerance by the combination of glybenclamide and GIP from results of two-way
ANOVA (Figs. 5A and 5B).
We examined the effects of the combination of glybenclamide and nateglinide on
glucose tolerance in the OGTT (Figs. 6A and 6B). Two-way ANOVA shows significant
main effects of both glybenclamide and nateglinide, and significant interaction between
their effects (Fig. 6B). The AUC values were almost the same for the groups treated
with glybenclamide alone, nateglinide alone or with both.
Figure 7 shows the changes of fasting blood glucose levels in normal rats that
were treated with E3024 (3 mg/kg) and/or glybenclamide (1 mg/kg) (Fig. 7A).
Two-way ANOVA indicated that there is a significant main effect of glybenclamide on
delta blood glucose values at 2 hr, whereas the main effect of E3024 was not significant
(Fig. 7B). There was no significant interaction. In the case of the combination of E3024
and nateglinide, a clear reduction in blood glucose levels was detected in both the
nateglinide alone, and the E3024 plus nateglinide groups (Fig. 8A). As for the
combination of E3024 and glybenclamide, two-way ANOVA demonstrated a
significant main effect of nateglinide on delta blood glucose values at 1 hr, whereas the
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main effect of E3024 was not significant (Fig. 8B). Neither was there a significant
interaction.
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Discussion
Takasaki et al. (2004) reported that a long-acting DPP-IV inhibitor, K579
((S)-1-[4-methyl-1-(2-pyrimidinyl)-4-piperidylamino]acetyl-2-pyrrolidinecarbonitrile),
significantly suppressed blood glucose elevation in glybenclamide-pretreated rats, but
they did not measure insulin concentrations. The insulinotropic effect of glybenclamide
was further amplified by intravenous infusion of GLP-1 in perfused rat pancreas, and in
NIDDM patients (Gutniak et al., 1996). Our study demonstrated that the combination of
glybenclamide and GLP-1 additively improved glucose tolerance in the OGTT in mice
fed a high-fat diet, and previously reported that E3024 elevated plasma active GLP-1
levels in the OGTT using Zucker fa/fa rats (Yasuda et al. 2006). Taken together these
findings suggest that DPP-IV inhibition may augment the glucose-lowering effects of
glybenclamide through the presence of active GLP-1 which has escaped from
inactivation by DPP-IV.
Glybenclamide binds to the sulfonylurea receptor type 1 (SUR1) of adenosine
triphosphate (ATP)-sensitive potassium (KATP) channels in pancreatic β-cell membranes,
and closes this channel. The resultant reduction in K+ permeability generates membrane
depolarization, opening of Ca2+ channels, elevation of [Ca2+]i, and eventually initiation
of Ca2+-dependent exocytosis of insulin-containing granules (Aguilar-Bryan et al.,
1995). Nateglinide also binds to SUR1, but another binding site, specific to nateglinide,
has been proposed (Fujita et al., 1996). On the other hand, GLP-1 binds to the GLP-1
receptor, a guanine nucleotide-binding protein-coupled receptor (GPCR), in pancreatic
β-cell membranes, and activates adenyl cyclase, leading to an increase in cAMP
production and activation of the cAMP-dependent protein kinase (protein kinase A;
PKA) pathway. This pathway is thought to potentiate glucose-induced insulin release.
In addition, the cAMP-GEFII·Rim pathway has been reported to be a PKA-independent
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mechanism for the potentiation of insulin secretion by GLP-1 (Kashima et al., 2001;
Gromada et al., 2004). Therefore, as the mechanisms of insulin secretion are different
for theses insulin secretagogues and GLP-1, synergistic or additive insulin release is
triggered with the combination of E3024 and glybenclamide or nateglinide.
GIP is the other incretin, released from K cells in the upper portion of the small
intestine after ingestion of glucose and fat (Yip and Wolfe, 2000). Like GLP-1,
GIP-(1-42), the active form of GIP, potentiates glucose-dependent insulin secretion, and
GIP-(1-42) is degraded by DPP-IV to inactive GIP-(3-42). Hansotia et al. (2004)
showed that DPP-IV inhibitors lowered glucose and increased plasma insulin in OGTT
in both GLP-1 receptor knockout mice and GIP receptor knockout mice. On the other
hand, the DPP-IV inhibitor actions were eliminated in double incretin receptor knockout
mice. Thus, both GLP-1 and GIP contribute to the improvement of glucose tolerance
elicited by DPP-IV inhibitors. Interestingly, both GLP-1 and GIP simulates insulin
secretion via GPCR activation of adenyl cyclase, coupled with the production of cAMP
(Ehses et al. 2002). The present study suggested that GIP is also be involved in the
combination effect of a DPP-IV inhibitor and an insulin secretagogue. Employing the
incretin receptor deficient mice and double knockout mice, we could document more
clearly and precisely the involvement of GLP-1 and/or GIP in the combination effect.
The combination of sulfonylures and nateglinide is not recommended, because
the “fast on-fast-off” mode of action of nateglinide is negated by the sustained action of
sulfonylureas (Van Gaal and De Leeuw, 2003). In this study, the combination of
glybenclamide and nateglinide did not show efficacy on glucose excursions in mice.
Also in in vitro study, simultaneous exposure of isolated rat islets to nateglinide and
glybenclamide did not cause a further increase of insulin release in comparison with that
produced by either agent alone (Ikenoue et al., 1997). Thus, the combination of
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glybenclamide and nateglinide may not be of use. However, E3024 could be combined
with either agent: the combination of E3024 and a sulfonylurea could reduce both
fasting and postprandial glucose levels, while that of E3024 and a rapid-acting insulin
secretagogue could enhance postprandial glucose-lowering effects.
We examined the combination of E3024 and glybenclamide or nateglinide on
fasting blood glucose levels in normal rats. E3024 alone did not reduce fasting blood
glucose levels, probably because E3024 induces insulin secretion in a
glucose-dependent fashion. Moreover, E3024 did not affect the blood-lowering effect of
either glybeclamide or nateglinide under fasting conditions. Therefore, the risk of
hypoglycemia may not be increased by combining E3024 and glybenclamide or
nateglinide, as compared with treatment using either glybenclamide or nateglinide alone,
despite the greater hypoglycemic effects seen for the combination compared with the
single treatment.
This is the first report on the statistically evident effectiveness of the combination
of E3024, a DPP-IV inhibitor, and glybenclamide, and the combination of E3024 and
nateglinide, presented with data for the accompanying changes in insulin levels. E3024
plus glybenclamide improved glucose tolerance additively, with insulin levels increased
synergistically in the OGTT in mice. Likewise, the combination of E3024 and
nateglinide ameliorated glucose tolerance additively with additively elevated insulin
levels. E3024 did not affect the glucose-lowering effect of glybenclamide or nateglinide
in fasting normal rats. These findings of our study suggest the combination of a DPP-IV
inhibitor and glybenclamide or nateglinide may be a promising option for the treatment
of type 2 diabetes, and especially for controlling postprandial hyperglycemia.
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Acknowledgements
We express thanks to S. Yoshikawa, K. Kira, E. Emori, F. Matsuura, H. Ikuta, Y.
Takeuchi, Y. Kitahara, M. Bando, and Y. Sakuma for assistance and suggestions.
Thanks are also due to I. Hishinuma and J. Nagakawa for valuable advice and
encouragement.
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Legends for figures
Figure 1. Chemical structure of E3024, 3-but-2-ynyl-5-methyl-2-piperazin-1-yl-3,5-
dihydro-4H- imidazo[4,5-d]pyridazin- 4-one tosylate.
Figure 2. Effects of a combination of E3024 (3 mg/kg) and glybenclamide (1 mg/kg) on
blood glucose (A and B) and plasma insulin levels (C) in an oral glucose tolerance
test using mice fed a high-fat diet. The compound(s) and glucose (2 g/kg) were
orally administered at -0.5 and 0 hr, respectively. Changes of blood glucose levels
and AUC values of delta blood glucose values between 0 and 2 hr are indicated in
A and B, respectively. Values are expressed as the mean ± S.E.M. of seven mice.
Results of two-way ANOVA on the AUC values and plasma insulin levels are
indicated in insets (B and C, respectively). Results of the Tukey multiple
comparison test are as follows: *, p<0.05 vs. vehicle group; #, p<0.05 vs. E3024
group; $, p<0.05 vs. glybenclamide group.
Figure 3. Effects of a combination of E3024 (3 mg/kg) and nateglinide (50 mg/kg) on
blood glucose (A and B) and plasma insulin levels (C) in an oral glucose tolerance
test using mice fed a high-fat diet. The compound(s) and glucose (2 g/kg) were
orally administered at -0.5 and 0 hr, respectively. Changes of blood glucose levels
and AUC values of delta blood glucose values between 0 and 2 hr are indicated in
A and B, respectively. Values are expressed as the mean ± S.E.M. of seven mice.
Results of two-way ANOVA on the AUC values and plasma insulin levels are
indicated in insets (B and C, respectively). Results of the Tukey multiple
comparison test are as follows: *, p<0.05 vs. vehicle group; #, p<0.05 vs. E3024
group; $, p<0.05 vs. nateglinide group.
Figure 4. Effects of a combination of glybenclamide (1 mg/kg) and GLP-1 (1 µg) on
blood glucose levels in and oral glucose tolerance test using mice fed a high-fat
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diet. Glybenclamide was orally administered at -0.5 hr, and glucose (2 g/kg; p.o.)
and GLP-1 (1 µg; i.p.) treatment were conducted at 0 hr. Changes of blood glucose
levels and AUC values of delta blood glucose values between 0 and 2 hr are
indicated. Values are expressed as the mean ± S.E.M. of six mice. Results of
two-way ANOVA on the AUC values are indicated in an inset. Results of the
Tukey multiple comparison test are as follows: *, p<0.05 vs. vehicle + saline
group; #, p<0.05 vs. vehicle + GLP-1 group; $, p<0.05 vs. glybenclamide + saline
group.
Figure 5. Effects of a combination of glybenclamide (1 mg/kg) and GIP (1 µg) on blood
glucose levels in and oral glucose tolerance test using mice fed a high-fat diet.
Glybenclamide was orally administered at -0.5 hr, and glucose (2 g/kg; p.o.) and
GIP (1 µg; i.p.) treatment were conducted at 0 hr. Changes of blood glucose levels
and AUC values of delta blood glucose values between 0 and 2 hr are indicated.
Values are expressed as the mean ± S.E.M. of six mice. Results of two-way
ANOVA on the AUC values are indicated in an inset. Results of the Tukey
multiple comparison test are as follows: *, p<0.05 vs. vehicle + saline group; #,
p<0.05 vs. vehicle + GIP group.
Figure 6. Effects of a combination of glybenclamide (1 mg/kg) and nateglinide (50
mg/kg) on blood glucose levels in and oral glucose tolerance test using mice fed a
high-fat diet. The compound(s) and glucose (2 g/kg) were orally administered at
-0.5 and 0 hr, respectively. Changes of blood glucose levels and AUC values of
delta blood glucose values between 0 and 2 hr are indicated. Values are expressed
as the mean ± S.E.M. of six mice. Results of two-way ANOVA on the AUC values
are indicated in an inset (B). Results of the Tukey multiple comparison test are as
follows: *, p<0.05 vs. vehicle group.
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Figure 7. Effects of a combination of E3024 (3 mg/kg) and glybenclamide (1 mg/kg) on
fasting blood glucose levels in normal rats. Changes of blood glucose levels (A)
and delta blood glucose values between 0 and 2 hr (B) are indicated. Results are
expressed as the mean ± S.E.M. of six rats. Results of two-way ANOVA on the
delta blood glucose levels at 2 hr are indicated in an inset (B). Results of the Tukey
multiple comparison test are as follows: *, p<0.05 vs. vehicle group; #, p<0.05 vs.
E3024 group.
Figure 8. Effects of a combination of E3024 (3 mg/kg) and nateglinide (50 mg/kg) on
fasting blood glucose levels in normal rats. Changes of blood glucose levels (A)
and delta blood glucose values between 0 and 1 hr (B) are indicated. Results are
expressed as the mean ± S.E.M. of six rats. Results of two-way ANOVA on the
delta blood glucose levels at 1 hr are indicated in an inset (B). Results of the Tukey
multiple comparison test are as follows: *, p<0.05 vs. vehicle group; #, p<0.05 vs.
E3024 group.
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