Comparison of the anti-diabetic effects of GIP- and GLP-1-receptor activation in obese diabetic (ob/ob) mice: studies with DPP IV resistant N-AcGIP and exendin(1-39)amide.

Abstract

The two major incretin hormones, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are being actively explored as anti-diabetic agents because they lower blood glucose through multiple mechanisms. The rapid inactivation of GIP and GLP-1 by the ubiquitous enzyme, dipeptidyl peptidase IV (DPP IV) makes their biological actions short-lived, but stable agonists such as N-acetylated GIP (N-AcGIP) and exendin(1-39)amide have been advocated as stable and specific GIP and GLP-1 analogues.
The present study examined the sub-chronic (14 days) anti-diabetic actions of single daily doses of N-AcGIP and exendin(1-39)amide given alone or in combination to obese diabetic (ob/ob) mice over a 14-day period.
Initial experiments confirmed the potent anti-hyperglycaemic and insulinotropic properties of N-AcGIP and exendin(1-39)amide. Sub-chronic administration of N-AcGIP alone or in combination with exendin(1-39)amide significantly decreased non-fasting plasma glucose and improved glucose tolerance compared to control ob/ob mice. This was associated with a significant enhancement of the insulin response to glucose and a notable improvement of insulin sensitivity. Combined treatment with N-AcGIP and exendin(1-39)amide also significantly decreased glycated haemoglobin. Exendin(1-39)amide alone had no significant effect on any of the metabolic parameters monitored. In addition, no significant effects were observed on body weight and food intake in any of the treatment groups.
The results illustrate significant anti-diabetic potential of N-AcGIP alone and in combination with exendin(1-39)amide.

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DIABETES/METABOLISM RESEARCH AND REVIEWS
Diabetes Metab Res Rev 2007; 23: 572–579.
Published online 21 February 2007 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/dmrr.729
RESEARCH ARTICLE
Comparison of the anti-diabetic effects of GIP- and
GLP-1-receptor activation in obese diabetic (ob/ob)
mice: studies with DPP IV resistant N-AcGIP and
exendin(1–39)amide
Nigel Irwin1*
Paula L. McClean1
Roslyn S. Cassidy1
Finbarr P. M. O’Harte1
Brian D. Green2
Victor A. Gault1
Patrick Harriott2
Peter R. Flatt1
1School of Biomedical Sciences,
University of Ulster, Coleraine,
Northern Ireland, UK
2School of Biology and Biochemistry,
Queen’s University of Belfast, Belfast,
Northern Ireland, UK
*Correspondence to: Nigel Irwin,
School of Biomedical Sciences,
University of Ulster, Coleraine,
Northern Ireland, BT52 1SA, UK.
E-mail: n.irwin@ulster.ac.uk
Received: 6 September 2006
Revised: 22 December 2006
Accepted: 2 January 2007
Abstract
Background
insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are
being actively explored as anti-diabetic agents because they lower blood
glucose through multiple mechanisms. The rapid inactivation of GIP and
GLP-1 by the ubiquitous enzyme, dipeptidyl peptidase IV (DPP IV) makes
their biological actions short-lived, but stable agonists such as N-acetylated
GIP (N-AcGIP) and exendin(1-39)amide have been advocated as stable and
specific GIP and GLP-1 analogues.
Thetwomajorincretinhormones,glucose-dependent
Methods
diabetic actions of single daily doses of N-AcGIP and exendin(1-39)amide
given alone or in combination to obese diabetic (ob/ob) mice over a 14-day
period.
The present study examined the sub-chronic (14 days) anti-
Results
insulinotropic properties of N-AcGIP and exendin(1-39)amide. Sub-chronic
administration of N-AcGIP alone or in combination with exendin(1-39)amide
significantly decreased non-fasting plasma glucose and improved glucose
tolerance compared to control ob/ob mice. This was associated with a
significant enhancement of the insulin response to glucose and a notable
improvement of insulin sensitivity. Combined treatment with N-AcGIP
and exendin(1-39)amide also significantly decreased glycated haemoglobin.
Exendin(1-39)amide alone had no significant effect on any of the metabolic
parameters monitored. In addition, no significant effects were observed on
body weight and food intake in any of the treatment groups.
Initial experiments confirmed the potent anti-hyperglycaemic and
Conclusions
AcGIP alone and in combination with exendin(1-39)amide. Copyright  2007
John Wiley & Sons, Ltd.
The results illustrate significant anti-diabetic potential of N-
Keywords
tidase IV (DPP IV); glucagon-like peptide-1 (GLP-1); analogue; exendin; glucose
homeostasis
glucose-dependent insulinotropic polypeptide (GIP); dipeptidylpep-
Copyright  2007 John Wiley & Sons, Ltd.

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Actions of GIP and GLP-1
573
Introduction
Glucose-dependent insulinotropic polypeptide (GIP) and
glucagon-like peptide-1 (GLP-1) are gastrointestinal
hormonesthatregulate post-prandialglucose homeostasis
[1].Theprimaryfunction
hormones is the glucose-dependent stimulation of insulin
release from the pancreatic beta-cells following nutrient
absorption [2]. However, recent significant advances
in the understanding of GIP and GLP-1 function have
shown that they possess a number of secondary activities
important for blood glucose regulation [3]. These actions
include up-regulation of insulin biosynthesis, stimulation
of glucose uptake and metabolism as well as beta-cell
proliferation, survival and growth [4,5]. Despite their
similarities, GIP and GLP-1 have several distinguishing
features. GLP-1 is known to decrease the feeding activity
and body weight and to inhibit gastric emptying and
glucagon secretion, whereas GIP has no such effects [3,6].
GIPandGLP-1aretranscribedonseparategenes[7,8]and
each possess its own specific G-protein coupled receptor
[9]. Recent evidence has shown that the signalling
pathways of GIP and GLP-1 are mechanistically different
[10].
These anti-diabetic features of GIP and GLP-1 provide
the basis for a recent upsurge of interest in the
pharmaceutical industry for exploitation as new therapies
[4,5]. Furthermore, since the insulin-releasing actions
of GIP and GLP-1 are glucose-dependent, unwanted
hypoglycaemic episodes are rare, providing a distinct
advantage over more traditional anti-diabetic drugs such
as the sulphonylureas [11]. However, the therapeutic
potential of GIP and GLP-1 is severely hindered through
rapid N-terminal degradation in the circulation by the
ubiquitous enzyme dipeptidyl peptidase IV (DPP IV). DPP
IV cleaves the N-terminal dipeptide of GIP and GLP-1
(Tyr1–Ala2in GIP and His1–Ala2in GLP-1), causing a
complete loss of insulinotropic action of these incretin
hormones [12].
Nonetheless, various strategies to overcome the rapid
degradation of GIP and GLP-1 are proving successful.
Several N-terminally modified analogues of GIP and
GLP-1 have been tested that are profoundly resistant
to DPP IV. For example, in GIP, modifications at Tyr1
appear to yield DPP IV resistant analogues with greater
bioactivity than Ala2or Glu3modifications, with N-
acetylated GIP (N-AcGIP) being most effective [13].
Interestingly, exendin(1–39)amide is a 39-amino acid
peptide isolated from the Gila monster salivary gland
that shares 53% sequence homology with GLP-1 and
acts as a specific DPP IV resistant agonist of the GLP-
1 receptor [14]. This peptide also termed exenatide
has been approved for the clinical treatment of type 2
diabetes in United States under the trade name Byetta
[15]. Thus, N-AcGIP and exendin(1–39)amide represent
DPP IV resistant GIP and GLP-1 agonists with enhanced
therapeutic potential.
ofthesetwoincretin
Although
enhanced anti-diabetic potential of various DPP IV resis-
tant analogues of GIP and GLP-1 [4–6], none have
evaluated the therapeutic potential of their combined
administration. The present study has directly compared
the anti-diabetic actions of a 14-day treatment of ob/ob
mice with N-AcGIP, exendin(1–39)amide or a combina-
tion of both peptides. Effects on basal plasma glucose
and insulin, glucose homeostasis, insulin sensitivity, gly-
cated haemoglobin and pancreatic insulin content were
examined. The results illustrate significant anti-diabetic
potential of N-AcGIP alone and in combination with
exendin(1–39)amide.
manystudieshave demonstrated the
Methods
Animals
Obese diabetic (ob/ob) mice derived from the colony
maintained at Aston University, UK, [16] were used at
15–19 weeks of age. Animals were housed in an air-
conditioned room at22 ± 2◦C witha 12-h light/12-h dark
cycle (08:00–20:00 h). Drinking water and standard
rodent maintenance diet (Trouw Nutrition, Cheshire,
UK) were freely available. All animal experiments
were carried out in accordance with the UK Animals
(Scientific Procedures) Act 1986. No adverse effects were
observed following a long-term administration of N-
AcGIP, exendin(1–39)amide or a combination of both
peptides.
Synthesis, purification and
characterization of GIP, N-AcGIP and
GLP-1
GIP, N-AcGIP and GLP-1 were sequentially synthesized
on an Applied Biosystems automated peptide synthe-
sizer (Model 432 A, Foster City, CA, USA) using a
standard solid-phase Fmoc peptide chemistry as pre-
viously reported [13]. For N-AcGIP, an acetyl adduct
was incorporated at the N-terminal Tyr1of native GIP.
Peptides were judged pure by reversed-phase HPLC
on a Waters Millenium 2010 chromatography system
(Software version 2.1.5) and subsequently characterized
using matrix-assisted laser desorption ionization-time of
flight mass spectrometry as described previously [17].
Exendin(1–39)amide was purchased from the American
Peptide Company (Sunnyvale, CA, USA) and character-
izedusingmatrix-assistedlaserdesorptionionization-time
of flight mass spectrometry.
In vitro insulin secretion
BRIN-BD11 cells were seeded into 24-multi-well plates at
a density of 1.0 × 105cells per well, and allowed to attach
overnight at 37◦C. Acute tests for insulin release were
Copyright  2007 John Wiley & Sons, Ltd.
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574
N. Irwin et al.
preceded by a 40-min pre-incubation at 37◦C in 1.0-mL
Krebs Ringer bicarbonate buffer (115 mM NaCl, 4.7 mM
KCl, 1.28 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4,
10 mM NaHCO3, 0.5% (w/v) BSA, pH 7.4) supplemented
with 1.1-mM glucose. Test incubations were performed
in the presence of 5.6-mM glucose with a range of
concentrations (10−12− 10−8M) of GIP, GLP-1, N-AcGIP
and exendin(1–39)amide. After a 20-min incubation, the
buffer was removed from each well and aliquots (200 µL)
were used for the measurement of insulin. The origin and
functional characteristics of BRIN BD11 cells are detailed
elsewhere [18].
Acute effects of GIP, GLP-1, N-AcGIP
and exendin(1–39)amide on
glucose-lowering and insulin release in
(ob/ob) mice
The effects of GIP, GLP-1, N-AcGIP and exendin(1–39)
amide on plasma glucose and insulin concentrations were
examined in an 18-h fasted obese diabetic (ob/ob) mice.
Ob/ob mice received intra-peritoneal injection of glucose
alone (18-mmol/kg body weight) or in combination with
GIP, GLP-1, N-AcGIP or exendin(1–39)amide (each at
25-nmol/kg body weight). Blood samples were collected
from the cut tip of the tail vein of a conscious mice
into the chilled fluoride/heparin coated glucose micro-
centrifuge tubes (Sarstedt, N¨ umbrecht, Germany) at the
times indicated in the Figures. The resulting plasma was
then aliquoted into fresh tubes and stored at −20◦C prior
to glucose and insulin determinations.
Sub-chronic effects of N-AcGIP,
exendin(1–39)amide and combined
peptide administration in ob/ob mice
Groups of ob/ob mice received once daily intra-peritoneal
injections (17:00 h) of either saline (0.9%, w/v, NaCl),
N-AcGIP, exendin(1–39)amide or a combination of
both peptides (all at 12.5 nmol/kg body weight/day).
This dose was chosen such that the combined peptide
administration corresponded to a net incretin dose of 25-
nmol/kg body weight as used in the acute tests. Previous
studies using GIP and GLP-1 analogues have shown to
exert in vivo effects within this dose range [19,20]. Food
intake and body weight were recorded daily from 4 days
before commencement of the treatment regimes. Plasma
glucose and insulin concentrations were monitored at
2–4-dayinterval(10:00h).OnDay14,groupsofanimals
were used to evaluate intra-peritoneal glucose tolerance
(18 mmol/kg) and insulin sensitivity (50 U/kg). Glycated
haemoglobin was also determined on Day 14. All acute
testswere commenced at10:00h.Allblood samples were
collected from the cut tip of the tail vein of a conscious
mice into the chilled fluoride/heparin coated glucose
micro-centrifuge tubes (Sarstedt, N¨ umbrecht, Germany)
at the times indicated in the Figures. Blood samples
–12
Log10 Peptide Concentration (M)
–11–10–9–8
0
1
2
3
Control
GIP
GLP-1
N-AcGIP
Exendin(1-39)amide
*
**
***
****
****
**
**
***
***
***
***
******
******
Insulin secretion
(ng/106 cells/20 min)
Figure 1. Insulin-releasing activity of GIP, GLP-1, N-AcGIP and
exendin(1–39)amide in the clonal pancreatic beta-cell line,
BRIN-BD11. After a pre-incubation (40 min), the effects of
various concentrations of peptide were tested on insulin release
during a 20-min incubation. Values are means ± SEM for
eight separate observations.∗p < 0.05,∗∗p < 0.01,∗∗∗p < 0.01
compared to 5.6-mM glucose control
were immediately centrifuged using a Beckman micro-
centrifuge (Beckman Instruments, Galway, Ireland) for
30 s at 13000 g. The resulting plasma was then aliquoted
into fresh tubes and stored at −20◦C prior to glucose and
insulin determinations.
Biochemical analyses
Plasma glucose was assayed by an automated glucose
oxidase procedure [13] using a Beckman Glucose
Analyser II. Glycated haemoglobin was measured using
a commercially available kit purchased from Chirus Ltd.
(Watford, UK) Insulin was assayed by a modified dextran-
charcoal RIA as described previously [13].
Statistics
Results are expressed as mean ± SEM. Data were
compared using repeated measures ANOVA or one-
way ANOVA, followed by the Student-Newman-Keuls
post hoc test. Incremental areas under plasma glucose
and insulin curves were calculated using a computer-
generated program employing the trapezoidal rule [13]
with baseline subtraction. Groups of data were considered
to be significantly different if p < 0.05.
Results
Stimulation of in vitroinsulin secretion
All
exendin(1–39)amide,
0.001) enhanced insulin release in a concentration-
dependent manner compared to 5.6-mM glucose control
(Figure 1). There was no significant difference in potency
between the four peptides. At the highest concentration
tested (10−8M), the peptides stimulated insulin secretion
by 2.1–2.4-fold compared to 5.6-mM glucose control.
four peptides;GIP,
significantly
GLP-1,
N-ACGIP
(p < 0.05 − p <
and
Copyright  2007 John Wiley & Sons, Ltd.
Diabetes Metab Res Rev 2007; 23: 572–579.
DOI: 10.1002/dmrr

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Actions of GIP and GLP-1
575
Acute anti-hyperglycaemic and insulin
releasing activity in ob/ob mice
The relative glucose-lowering abilities of GIP, GLP-1,
N-AcGIP and exendin(1–39)amide (25-nmol/kg body
weight) in ob/ob mice are shown in Figure 2(A). Injection
of glucose alone resulted in a rapid and marked
increase in plasma glucose that continued to rise until
60 min. Native GIP had a tendency to reduce glucose
concentrations, and by 60 min, levels were significantly
(p < 0.05) decreased compared to control (Figure 2(A)).
Similarly, N-AcGIP, GLP-1 and exendin(1–39)amide
reduced plasma glucose concentrations compared to
control with significantly (p < 0.01 − p < 0.001) reduced
glucose levels between 30and60 min.The overallglucose
areaunderthecurve valuesweresignificantly(p < 0.05 −
p < 0.001) reduced in all four peptide treated groups
compared to control, with N-AcGIP being significantly
(p < 0.05) more effective than native GIP. As shown
in Figure 2(B), these anti-hyperglycaemic effects were
linked to corresponding changes in plasma insulin. All
four peptides significantly increased glucose-stimulated
insulin concentrations between 15 and 60 min. Native
GIP caused a significantly greater (p < 0.05) overall
insulin response compared to glucose alone. N-AcGIP
was significantly more potent than native GIP (p < 0.01).
GLP-1 and exendin(1–39)amide had similar enhanced
insulinotropic effects, evoking significantly (p < 0.01 and
p < 0.001; respectively) greater overall insulin release
compared to control (Figure 2(B)).
Sub-chronic effects of N-AcGIP,
exendin(1–39)amide and a
combination of both peptides on
non-fasting plasma glucose and insulin
levels and glycated haemoglobin
concentrations
Administration of N-AcGIP, exendin(1–39)amide or a
combination of both peptides had no effect on food
intake or body weight (data not shown). Plasma glucose
concentrations were progressively reduced, resulting in
significantly (p < 0.05) lowered glucose concentrations
at 14 days in the groups treated with N-AcGIP alone
or in combination with exendin(1–39)amide (Figure 3).
Glycated haemoglobin concentrations were significantly
(p < 0.05) reduced in the combined N-AcGIP and
exendin(1–39)amide treated group (Figure 3). These
changes were accompanied by a tendency towards
elevated insulin concentrations, but these did not achieve
a statistical significance over the study period (Figure 3).
No differences onplasma glucose orinsulin were observed
in exendin(1–39)amide treated mice alone (Figure 3).
0 15 304560
10
20
30
40
50
Glucose alone
Glu + GIP
Glu + GLP-1
Glu + N-AcGIP
Glu + Exendin(1-39)amide
*
**
***
***
***
**
**
Time (min)
Plasma glucose
(mmol/l)
0
500
1000
1500
Control
GIP
GLP-1
N-AcGIP
Exendin(1-39)amide
*
**
***
∆
∆∆
***
***
Plasma glucose AUC
(mmol/l.min)
0 153045 60
0
5
10
15
20
25
Glucose alone
Glu + GIP
Glu + GLP-1
***
***
Glu + N-AcGIP
Glu + Exendin(1-39)amide
***
*
*
*
*
****
*
Time (min)
Plasma insulin
(ng/ml)
0
250
500
750
Control
GIP
GLP-1
N-AcGIP
Exendin(1-39)amide
*
**
***
Plasma insulin AUC
(ng/ml.min)
AB
Figure 2. Acute glucose lowering and insulin releasing activity of GIP, GLP-1, N-AcGIP and exendin(1–39)amide in an 18-h fasted
(ob/ob) mice. Plasma glucose and insulin concentrations were measured prior to and after intra-peritoneal administration of
glucose alone (18 mmol/kg) as a control, or in combination with peptide (25 nmol/kg). The glucose and insulin area under
the curve (AUC) between 0 and 60 min are shown in bottom panels. Values represent means ± SEM for eight mice.∗p < 0.05,
∗∗p < 0.01,∗∗∗p < 0.01 compared to glucose alone.?p < 0.05,??p < 0.01 compared to native GIP
Copyright  2007 John Wiley & Sons, Ltd.
Diabetes Metab Res Rev 2007; 23: 572–579.
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N. Irwin et al.
–4048 1216
15
25
35
Saline
Exendin(1-39)amide
N-AcGIP
Exendin(1-39)amide + N-AcGIP
Time (days)
Plasma glucose
(mmol/l)
*
*
–40481216
20
40
60
80
Saline
Exendin(1-39)amide
N-AcGIP
Exendin(1-39)amide + N-AcGIP
Time (days)
Plasma insulin
(ng/ml)
0
5
10
15
Saline
Exendin(9-39)amide
N-AcGIP
Exendin(9-39)amide + N-AcGIP
*
%HbA1C
C
A
B
Figure 3. Effects of daily N-AcGIP, exendin(1–39)amide or
combined peptide administration on plasma glucose (A), insulin
(B) and glycated haemoglobin concentrations (C). N-AcGIP,
exendin(1–39)amide, a combination of both peptides (each at
12.5 nmol/kg/day) or saline (control) were administered for
14 days as indicated by the horizontal black bar. Values are
mean ± SEM for eight mice.∗p < 0.05 compared to control
Sub-chronic effects of N-AcGIP,
exendin(1–39)amide and a
combination of both peptides on
glucose tolerance
Treatment with N-AcGIP alone or in combination with
exendin(1–39)amide for 14 days resulted in a significant
improvement in glucose tolerance and glucose-mediated
insulin release. N-AcGIP produced a 27% reduction
in the overall glycaemic excursion (p < 0.05). This
was accompanied by a 194% increase in the overall
insulin release (p < 0.05) (Figure 4). When N-AcGIP
administration was combined with exendin(1–39)amide,
a similar effect was noted (p < 0.05; in both cases)
(Figure 4). However, exendin(1–39)amide treatment
alone had no significant effects on glucose stimulated
insulin release or glycaemic excursion (Figure 4).
Sub-chronic effects of N-AcGIP,
exendin(1–39)amide and a
combination of both peptides on
insulin sensitivity
As shown in Figure 5, the hypoglycaemic action of
insulin was significantly augmented in terms and area
under the curve measures in ob/ob mice treated for
14 days with N-AcGIP (1.3-fold; p < 0.05) alone or
combinedwithexendin(1–39)amide(1.5-fold;p < 0.01).
Treatment for 14 days with exendin(1–39)amide alone
had no significant effects on the glucose lowering effects
of exogenous insulin in ob/ob mice (Figure 5).
Discussion
Previous studies have shown that daily administration of
exendin(1–39)amide or stable GIP analogues, including
N-AcGIP result in a significant amelioration of diabetes in
type 2 animal models [19,21–23]. Direct comparison of
theanti-diabetic properties ofthese stable incretinpeptide
analogues, and evaluation of the possible effects of com-
bined treatment, have not been carried out. The present
study examined the effects of sub-chronic administration
of N-AcGIP, exendin(1–39)amide or a combination of
both peptides in adult ob/ob mice. In harmony with pre-
vious findings, acute studies in ob/ob mice confirmed the
potent anti-hyperglycaemic and insulinotropic properties
of N-AcGIP and exendin(1–39)amide [13,24].
These observations of unchanged body weight, food
intake and non-fasting plasma insulin concentrations
are broadly similar to the previously reported long-term
effects of stable GIP agonists in this animal model, thereby
confirming a spectrum of useful anti-diabetic effects
[20,23]. Combination of N-AcGIP with an equal dose of
exendin(1–39)amide did not result in appreciably greater
effects than observed with N-AcGIP alone. However, a
small decrease of glycated haemoglobin was observed
over a short 14-day study period in this group, suggesting
a potential synergistic action. This effect may have been
magnified, if study was carried out over a longer time
course.
Perhaps, the greatest surprise from the present study
was the lack of effect of exendin(1–39)amide when
given alone by daily injection over 14 days to ob/ob
mice. This contrasts sharply with similar studies in db/db
mice, fa/fa rats and Zucker rats, which showed that
long-term administration of exendin(1–39)amide low-
ered blood glucose and HbA1c, significantly reduced food
intake causing weight loss and improved insulin sensi-
tivity, while insulin concentrations were also reported
to be higher [21,22,25]. Importantly, the batch of
exendin(1–39)amide used in the present study was of
Copyright  2007 John Wiley & Sons, Ltd.
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Actions of GIP and GLP-1
577
0 1530 4560
10
30
50
70
*
**
*
*
*
Exendin(1-39)amide + N-AcGIP
Saline
Exendin(1-39)amide
N-AcGIP
Time (min)
Plasma glucose
(mmol/l)
0153045 60
20
40
60
80
Saline
Exendin(1-39)amide
N-AcGIP
Exendin(1-39)amide + N-AcGIP
Time (min)
Plasma insulin
(ng/ml)
0
1000
2000
Saline
Exendin(9-39)amide
N-AcGIP
Exendin(9-39)amide + N-AcGIP
*
*
Plasma glucose
(mmol/l.min)
0
400
800
1200
*
*
Saline
Exendin(1-39)amide
N-AcGIP
Exendin(1-39)amide + N-AcGIP
Plasma insulin
(ng/ml.min)
BA
Figure 4. Effects of daily N-AcGIP, exendin(1–39)amide and combined peptide administration on glucose tolerance (A) and
plasma insulin response to glucose (B). Tests were conducted after 14 daily injections of either N-AcGIP, exendin(1–39)amide,
a combination of both peptides (each at 12.5 nmol/kg/day) or saline (control). Glucose (18 mmol/kg) was administered by
intra-peritoneal injection at the time indicated by the arrow. Plasma glucose and insulin AUC values for a 0–60 min post-injection,
are shown in the bottom panels. Values are mean ± SEM for eight mice.∗p < 0.05 and∗∗p < 0.01 compared to control
0 15 30 4560
20
40
60
80
100
Saline
Exendin(1-39)amide
N-AcGIP
Exendin(1-39)amide + N-AcGIP
Time (min)
Plasma glucose
(% of basal)
0
500
1000
1500
Saline
Exendin(1-39)amide
N-AcGIP
Exendin(1-39)amide + N-AcGIP
**
*
Plasma glucose AUC
(% of basal.min)
Figure 5. Effects of daily N-AcGIP, exendin(1–39)amide or combined peptide administration on insulin sensitivity. Tests were
conducted after 14 daily injections of either N-AcGIP, exendin(1–39)amide, a combination of both peptides (each at 12.5
nmol/kg/day) or saline (control). Insulin (50 U/kg) was administered by intra-peritoneal injection at the time indicated by the
arrow. Plasma glucose AUC values for a 0–60 min post–injection, are shown in the bottom panel. Values are mean ± SEM for eight
mice.∗p < 0.05 and∗∗p < 0.01 compared to control
confirmed molecular identity and proven to be active
in both in vitro insulin secretion tests and acute studies
in ob/ob mice. Indeed, results of the latter correspond
with similarly published acute studies in this animal
model [21]. The most plausible explanation for the
lack of such chronic effects of exendin(1–39)amide,
Copyright  2007 John Wiley & Sons, Ltd.
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N. Irwin et al.
therefore, lies with either the dose or possible GLP-1
receptor de-sensitization. It seems unlikely to us that
a reduced dose of 12.5 nmol/kg would remove an
effect of exendin(1–39)amide, whereas potency of N-
AcGIP is retained. Furthermore, other studies testing
exendin(1–39)amide in animal models have employed
doses of 1 [26], 3 [27], 24 [22] or 50 nmol/kg [21].
The alternative that GLP-1 receptor de-sensitization lies
behind the present poor long-term responses in ob/ob
mice is perhaps more plausible because such a phe-
nomenon has been observed previously with GLP-1
in vitro [28], although not to any appreciable extent
in vivo [29]. However, this effect would appear to be
species and animal model specific. Also, the disappoint-
ing sub-chronic effects of exendin(1–39)amide observed
here contrast sharply with the prominent anti-diabetic
actions of the stable agonist (Val8)GLP-1 administered at
25 nmol/kg to ob/ob mice [19]. Thus, further studies are
required to resolve this issue, which seems to stem from
issues surrounding chemical nature of the GLP-1 mimetic,
dose and animal model employed. Determination of the
pharmacokinetics of exendin(1–39)amide in ob/ob mice
would also be useful, especially in relation to the time of
administering glucose load.
In conclusion, the present study indicates that once
daily injection of N-AcGIP is an effective means
of improving diabetes control in ob/ob mice. Some
evidenceforbenefitof
exendin(1–39)amide was obtained, but this GLP-1
mimetic is considerably less effective than N-AcGIP or
(Val8)GLP-1 [19] in this commonly employed diabetic
animal model.
combinedtreatmentwith
Acknowledgements
These studies were supported by a grant from the Diabetes UK
and the University of Ulster Research Strategy Funding. The
authors would also like to acknowledge Dr C. J. Bailey for his
kind donation of ob/+ mice breeding pairs.
References
1. Creutzfeldt W.
diabetes–incretins as therapeutic agents. Exp Clin Endocrinol
Diabetes 2001; 109: S288–S303.
2. Nauck MA,Bartels E,Orskov C,Ebert R,Creutzfeldt W.Additive
insulinotropic effects of exogenous synthetic human gastric
inhibitory polypeptide and glucagon-like peptide-1-(7–36)
amide infused at near-physiological insulinotropic hormone
and glucose concentrations. J Clin Endocrinol Metab 1993; 76:
912–917.
3. Gault VA, O’Harte FPM, Flatt PR. Glucose-dependent insuli-
notropic polypeptide (GIP): anti-diabetic and anti-obesity
potential? Neuropeptides 2003; 37: 253–263.
4. Meier JJ, Gallwitz B, Siepmann N, et al. Gastric inhibitory
polypeptide(GIP) dose-dependently
secretioninhealthyhumansubjectsateuglycaemia. Diabetologia
2003; 46: 798–801.
5. Green BD, Gault VA, O’Harte FP, Flatt PR. Structurally modified
analogues of glucagon-like peptide-1 (GLP-1) and glucose-
dependent insulinotropicpolypeptide
antidiabetic agents. Curr Pharm Des 2004; 10: 3651–3662.
Theentero-insular axis in type2
stimulates glucagon
(GIP) asfuture
6. Meier JJ, Nauck MA, Schmidt WE, Gallwitz B. Gastric inhibitory
polypeptide: the neglected incretin revisited. Regul Pept 2002;
107: 1–13.
7. Mojsov S,Heinrich G, Wilson IB,
Habener JF. Preproglucagon gene expression in pancreas and
intestine diversifies at the level of post-translational processing.
J Biol Chem 1986; 261: 11880–11889.
8. Inagaki N, Seino Y, Takeda J,
polypeptide: structure and chromosomal localization of the
human gene. Mol Endocrinol 1989; 3: 1014–1021.
9. Moens K, Heimberg H,Flamez D,
functional activity of glucagon, glucagon-like peptide I, and
glucose-dependent insulinotropic peptide receptors in rat
pancreatic islet cells. Diabetes 1996; 45: 257–261.
10. Miki T, Minami K,Shinozaki H,
glucose-dependent insulinotropic polypeptide and glucagon-like
peptide-1 on insulin secretion and gut motility. Diabetes 2005;
54: 1056–1063.
11. Nattrass M, Bailey CJ. New agents for Type 2 diabetes. Baillieres
Best Pract Res Clin Endocrinol Metab 1999; 13: 309–329.
12. Deacon CF. Circulationand degradation ofGIP and GLP-1.Horm
Metab Res 2004; 36: 761–765.
13. O’Harte FPM, Gault VA, Parker JC, et al. Improved stability,
insulin-releasing activity and antidiabetic potential of two novel
N-terminal analogues of gastric inhibitory polypeptide: N-acetyl-
GIP and pGlu-GIP. Diabetologia 2002; 45: 1281–1291.
14. Goke R, Fehmann HC, Linn T, et al. Exendin-4 is a high potency
agonist and truncated exendin-(9–39)-amide an antagonist at
the glucagon-like peptide 1-(7–36)-amide receptor of insulin-
secreting beta-cells. J Biol Chem 1993; 268: 19650–19655.
15. Triplitt C, Chiquette E. Exenatide: from the Gila monster to the
pharmacy. J Am Pharm Assoc 2006; 46: 44–52.
16. Bailey CJ, Flatt PR. Influence of genetic background and age on
the expression of the obese hyperglycemic syndrome in Aston
ob/ob mice. Int J Obes 1982; 6: 11–21.
17. Gault VA, Irwin N, Harriott P, Flatt PR, O’Harte FPM. DPP IV
resistance and insulin releasing activity of a novel di-substituted
analogue of glucose-dependent insulinotropic polypeptide,
(Ser2-Asp13)GIP. Cell Biol Int 2002; 27: 41–46.
18. McClenaghan NH,Barnett CR,Ah-Sing E,et al.Characterization
of a novel glucose-responsive insulin-secreting cell line,
BRIN-BD11, produced by electrofusion. Diabetes 1996; 45:
1132–1140.
19. Green BD, Lavery KS,Irwin N,
peptide-1 (GLP-1) analog (Val8)GLP-1 results in significant
improvements of glucose tolerance and pancreatic beta-cell
function after 3-week daily administration in obese diabetic
(ob/ob) mice. J Pharmacol Exp Ther 2006; 318: 914–921.
20. Irwin N, Gault VA, Green BD, et al. Antidiabetic potential of two
novel fatty acid derivatised, N-terminally modified analogues
of glucose-dependent insulinotropic polypeptide (GIP): N-
AcGIP(LysPAL16)andN-AcGIP(LysPAL37).BiolChem2005;386:
679–687.
21. Young AA, Gedulin BR, Bhavsar S, et al. Glucose-lowering and
insulin-sensitizing actions of exendin-4: studies in obese
diabetic (ob/ob, db/db) mice, diabetic fatty Zucker rats, and
diabetic rhesus monkeys (Macaca mulatta). Diabetes 1999; 48:
1026–1034.
22. Greig NH, Holloway HW, De Ore KA, et al. Once daily injection
of exendin-4 to diabetic mice achieves long-term beneficial
effects on blood glucose concentrations. Diabetologia 1999; 42:
45–50.
23. Irwin N,Clarke GC, Green BD,
antidiabetic activity of DPP IV resistant N-terminally modified
versus mid-chain acylated analogues of glucose-dependent
insulinotropic polypeptide. Biochem Pharmacol 2006; 72:
719–728.
24. Green BD,Mooney MH, Gault VA,
substitution in glucagon-like peptide-1(7–36)amide confers
dipeptidylpeptidase IV resistance with cellular and metabolic
actions similar to those of established antagonists glucagon-like
peptide-1(9–36)amide and exendin (9–39). Metabolism 2004;
53: 252–259.
25. Al-Barazanji KA, Arch JR, Buckingham RE,Tadayyon M. Central
exendin-4 infusion reduces body weight without altering plasma
leptin in (fa/fa) Zucker rats. Obes Res 2000; 8: 317–323.
26. Wang Q, Brubaker PL. Glucagon-like peptide-1 treatment delays
the onset of diabetes in 8 week-old db/db mice. Diabetologia
2002; 45: 1263–1273.
Ravazzola M,Orci L,
et al.Gastric inhibitory
et al. Expressionand
et al.Distinct effects of
et al. Novel glucagon-like
et al.Evaluationofthe
et al. Lys9
forGlu9
Copyright  2007 John Wiley & Sons, Ltd.
Diabetes Metab Res Rev 2007; 23: 572–579.
DOI: 10.1002/dmrr

Page 8

Actions of GIP and GLP-1
579
27. Szayna M, Doyle ME, Betkey JA, et al. Exendin-4 decelerates
food intake, weight gain, and fat deposition in Zucker rats.
Endocrinology 2000; 141: 1936–1941.
28. Delmeire D, Flamez D, Moens K, et al. Prior in vitro exposure to
GLP-1 with or without GIP can influence the subsequent beta
cell responsiveness. Biochem Pharmacol 2004; 68: 33–39.
29. Baggio LL, Kim JG, Drucker DJ. Chronic exposure to GLP-1R
agonists promotes homologous GLP-1 receptor desensitization
in vitro but does not attenuate GLP-1R-dependent glucose
homeostasis in vivo. Diabetes 2004; 53: S205–S214.
Copyright  2007 John Wiley & Sons, Ltd.
Diabetes Metab Res Rev 2007; 23: 572–579.
DOI: 10.1002/dmrr