Endocrinology Vol. 142, No. 10 4462-4468
Copyright © 2001 by The Endocrine Society
Insertion of an N-Terminal 6-Aminohexanoic Acid after the 7 Amino Acid Position of Glucagon-Like Peptide-1 Produces a Long-Acting Hypoglycemic Agent
Máire E. Doyle,
Nigel H. Greig,
Harold W. Holloway,
Jennifer A. Betkey,
Michel Bernier and
Josephine M. Egan
Diabetes Section (M.E.D., J.A.B., M.B., J.M.E.) and Drug
Design and Development Section (H.W.H., N.H.G.), National Institute on
Aging, National Institutes of Health, Baltimore, Maryland 21224
Address all correspondence and requests for reprints to: Dr. Josephine M. Egan, Diabetes Section 23, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, Maryland 21224. E-mail: eganj{at}vax.grc.nia.nih.gov
 |
Abstract
|
|---|
The use of glucagon-like peptide-1 (GLP-1) as a routine treatment
for type 2 diabetes mellitus is undermined by its short biological
half-life. A cause of degradation is its cleavage at the N-terminal HAE
sequence by the enzyme dipeptidyl peptidase IV (DPP IV). To
protect from DPP IV, we have studied the biological activity of a
GLP-1 analog in which 6-aminohexanoic acid (Aha) is
inserted between histidine and alanine at positions 7 and 8. We have
compared the biological activity of this new compound, GLP-1
Aha8, with the previously described GLP-1 8-glycine
(GLP-1 Gly8) analog. GLP-1
Aha8 (10 nM) was equipotent with GLP-1
(10 nM) in stimulating insulin secretion in RIN 1046-38
cells. As with GLP-1 Gly8, the binding affinity
of GLP-1 Aha8 for the GLP-1 receptor in intact
Chinese hamster ovary (CHO) cells expressing the human GLP-1 receptor
(CHO/GLP-1R cells) was reduced (IC50: GLP-1, 3.7
± 0.2 nM; GLP-1 Gly8, 41 ± 9
nM; GLP-1 Aha8, 22 ± 7
nM). GLP-1 Aha8 was also shown to
stimulate intracellular cAMP production 4-fold above basal at
concentrations as low as 0.5 nM. However, it exhibited a
higher ED50 when compared to GLP-1 and GLP-1
Gly8 (ED50: GLP-1, 0.036 ±
0.002 nM, GLP-1 Gly8, 0.13 ± 0.02
nM, GLP-1 Aha8, 0.58 ± 0.03
nM). A series of D-amino acid-substituted GLP-1
compounds were also examined to assess the importance of putative
peptidase-sensitive cleavage sites present in the GLP-1 molecule.
They had poor binding affinity for the GLP-1 receptor, and none of
these compounds stimulated the production of intracellular cAMP in
CHO/GLP-1R cells or insulin secretion in RIN 1046-38 cells. GLP-1
Aha8 (24 nmol/kg) administered sc to fasted Zucker
(fa/fa) rats (mean blood glucose, 195 ± 32 mg/dl)
lowered blood glucose levels to a nadir of 109 ± 3 mg/dl, and it
remained significantly lower for 8 h. Matrix-assisted linear
desorption ionization-time of flight mass spectrometry of GLP-1
Aha8 incubated with DPP IV (37 C, 2 h)
did not exhibit an N-terminal degradation product. Taken together,
these results show that insertion of Aha after the 7 position in GLP-1
produces an effective, long-acting GLP-1 analog, which may be useful in
the treatment of type 2 diabetes mellitus.
|
Introduction
|
|---|
GLUCAGON-LIKE PEPTIDE-1
(GLP-1)-(7–36)amide and GLP-1-(7–37) are
currently under review as treatments for type 2 diabetes mellitus
(1). Both are potent insulin secretagogues that are
synthesized and secreted by the L-cells of the intestine.
They are processed from proglucagon by cleavage of the
GLP-1-(1–37) form that itself is not an insulin
secretagogue. The majority of circulating GLP-1 in humans
is in the GLP-1-(7–36)amide form (2). When
given exogenously, it lowers blood glucose levels in type 2 diabetic
and nondiabetic subjects, primarily by stimulating insulin secretion in
a glucose-dependent manner. Despite its promise, GLP-1 is
unlikely to become a routinely used therapeutic agent because it is
rapidly degraded in vivo (3). The N-terminal
sequence of the peptide is readily cleaved within minutes, between the
alanine and glutamic acid (in the 8 and 9 positions, respectively) by
the enzyme, dipeptidyl peptidase IV (DPP IV) (4).
There have been several reports of GLP-1 homologs with
N-terminal modifications ranging from substitution of the alanine with
threonine, serine, 
-aminoisobutyric acid (5) or glycine
(5, 6) as well as glycation of the alanine
(7). To protect against cleavage by DPP IV we studied the
biological activity of a novel GLP-1 analog in which
6-aminohexanoic acid (Aha) is inserted between the histidine and
alanine at positions 7 and 8. We compare this new compound
(GLP-1 Aha8) with GLP-1
8-glycine (GLP-1 Gly8) analog in
terms of biological activity in vitro and in
vivo. We also examined the effect of the inclusion of 4 and 8 Aha
moieties [GLP-1
(Aha9)4 and
GLP-1 (Aha9)8,
respectively] in between the alanine and the glutamic acid at
positions 8 and 9 to examine the effects of the perturbance of the
GLP-1 structure in the N-terminal region.
As GLP-1 also contains substrate sites throughout its
sequence for several endonucleases (8), we examined the
potency of a series of GLP-1 analogs with substitution of
the D-configuration in the first 3, 5, 8, 12, and 21 amino
acids (from the N-terminal end) and culminating in a complete
D-amino acid GLP-1 sequence.
|
Materials and Methods
|
|---|
Peptides
Peptides were synthesized on a polyethylene
glycol-polystyrene resin using F-moc derivatives of amino acids in a
PerSeptive Biosystems (Cambridge, MA) automated peptide synthesizer
using piperidine-dimethyl formamide for deprotection and the
coupling reagent
2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate and 1-hydroxybenzotriazole for
coupling. The finished peptides were cleaved from the resin using
trifluoroacetic acid, precipitated with ether, and subjected to
purification using reverse phase HPLC on a C18
hydrophobic resin in 0.1% trifluoroacetic acid using an acetonitrile
gradient. The purity of the final material was verified using reverse
phase HPLC, and the mass of the peptide was verified using mass
spectrometry. All peptides were of 95% or greater purity. Table 1
shows the sequences of the
GLP-1 analogs studied.
Chemicals and cell lines
Isobutylmethylxanthine (IBMX) was purchased from
Calbiochem (La Jolla, CA). Exendin-4, exendin-(9–39), and
GLP-1-(7–36)amide were obtained from Bachem
(Torrance, CA). The cloned rat insulinoma cell line RIN 1046-38 was a
gift from Dr. Samuel A. Clark (Bio Hybrid Technologies, Shrewsbury, MA)
and was cultured as described previously (9). Chinese
hamster ovary (CHO) cells stably transfected with the human
GLP-1 receptor, CHO/GLP-1R cells, were previously
described (10).
Insulin secretion in vitro
RIN 1046-38 cells grown on 12-well plates that had reached
50–60% confluence were washed in glucose-free insulin secretion
buffer (9), followed by two 30-min preincubation periods
in fresh insulin secretion assay buffer (1 ml). The buffer was
replaced, and the cells were incubated with the peptides and glucose (5
mM) for 1 h at 37 C. The supernatant was then
collected and saved for determination of insulin content by enzyme
immunoassay (Crystal Chem, Chicago, IL). Cells were lysed by the
addition of hydrochloric acid (300 µl, 0.1 M, 20 min,
room temperature), and the protein content was quantified using the
Bradford method.
Intracellular cAMP determination
CHO/GLP-1R cells grown to 60–70% confluence on 12-well plates
were washed three times with Krebs-Ringer phosphate buffer (KRP) and
incubated with 1 ml KRP containing 0.1% BSA for 2 h at 37 C in a
humidified air incubator. Cells were then incubated in 1 ml KRP
supplemented with 0.1% BSA with IBMX (1 mM) in the
presence or absence of the peptides under study. The reaction was
stopped 30 min later by washing the intact cells three times with
ice-cold PBS. The intracellular cAMP was extracted by incubating the
cells in ice-cold perchloric acid (0.6 M, 1 ml, 5 min).
After adjusting the pH of the samples to 7 using potassium carbonate (5
M, 84 µl), sample tubes were vortexed, and the
precipitate formed was sedimented by centrifugation (5 min, 2000
x g, 4 C). The supernatant was vacuum-dried and
solubilized in 0.05 M Tris (pH 7.5) containing 4
mM EDTA (300 µl). Sodium carbonate (0.15
µM) and zinc sulfate (0.15 µM) were added
to the samples, which were then incubated for 15 min on ice. The
resulting salt precipitate was removed by centrifugation (5 min,
2000 x g, 4 C). The samples were assayed in
duplicate aliquots (50 µl) using a [3H]cAMP competitive
binding assay kit (Amersham Pharmacia Biotech, Little
Chalfont, UK).
Competitive binding of peptides to GLP-1 receptor
in intact cells
Binding studies were performed as described by
Montrose-Rafizadeh et al. (10). Briefly
CHO/GLP-1R cells were grown to confluence on 12-well plates and washed
with serum-free Ham’s F-12 medium for 2 h before the experiment.
After two washes in 0.5 ml binding buffer (10), cells were
incubated overnight at 4 C with 0.5 ml buffer containing 2% BSA, 17
mg/liter diprotin A (Bachem, Torrance, CA), 10
mM glucose, 1–1,000 nM
GLP-1 or other peptides, and 30,000 cpm
125I-GLP-1 (Amersham Pharmacia Biotech). At the end of the incubation the supernatant
was discarded, and the cells were washed three times with ice-cold PBS
and incubated at room temperature with 0.5 ml 0.5
N NaOH and 0.1% SDS for 10 min. Radioactivity in
cell lysates was measured in an Apec-Series -counter (ICN Biomedicals, Inc., Costa Mesa, CA). Specific binding was
determined as total binding minus the radioactivity associated with
cells incubated in the presence of a large excess of unlabeled
GLP-1 (1 µM).
Matrix-assisted linear desorption ionization (MALDI) mass
spectroscopy
GLP-1 (2 µM) and GLP-1
Aha8 (2 µM) were incubated with 5
mU recombinant DPP IV (Calbiochem, La Jolla, CA) in PBS
for 10 min and 2 h, respectively, at 37 C. Both compounds (100
µM and 100 µl) were incubated in an equivalent volume
of human serum at 37 C for 2 h. In all cases enzymatic reactions
were quenched by the addition of trifluoroacetic acid (0.1%, vol/vol,
final concentration). Samples were immediately analyzed using
MALDI-time of flight (MALDI-TOF) mass spectrometry. A Micromass
MALDI-TOF (Micromass, Beverly, MA) reflectron instrument was used at a
laser energy of 15–25% over a mass range of 1000–6000 Da, with five
laser shots summed per spectrum. 
-Cyano-4-hydroxycinnamic acid
(Sigma, St. Louis, MO) was used as a matrix and was
prepared to a concentration of 10 mg/ml in an 8 mg/ml ammonium
carbonate (Sigma) buffer. One-microliter samples were
diluted 50:50 (vol/vol) with matrix before being transferred to the
MALDI plate.
Determination of biological activity of GLP-1 Aha8 in
vivo
Six-month-old male Zucker fa/fa rats (Harlan,
Indianapolis, IN) and 6-month-old Wistar rats were used in this study.
Animals were cared for in accordance with protocols approved by the
animal care and use committee of the Gerontology Research Center, NIA
(Baltimore, MD). They were allowed ad libitum access to chow
and water and were on a 12-h light, 12-h dark cycle (lights on at
0700 h). The bedding for the Zucker rats was a paper-based
product, Carefresh (Absorption Co., Belingham, WA). The Zucker rats
were fasted on wire, in the absence of bedding, overnight before the
experiment. Wistar rats were fasted on their normal bedding. General
anesthesia was induced by an ip injection of pentobarbital (50 mg/kg).
A cannula was placed in the femoral artery for blood sampling, and the
peptides (GLP-1 Aha8 and
GLP-1 Gly8, 24 nmol/kg) were
injected sc into the nape of the animals’ necks (n = 5 for each
treatment group). Blood glucose levels were measured by the glucose
oxidase method using a Glucometer Elite (Bayer Corp.,
Tarrytown, NY).
Statistical analysis
All values are shown as the mean ± SEM, and
the differences among the groups were analyzed using ANOVA. The curves
for Figs. 3 and 4 were fitted with a four-parameter sigmoid logistic
regression equation using an iterative computer program
(11), and the EC50 and
IC50 values in Table 2 were calculated from the fitted
data.
View larger version (24K):
[in this window]
[in a new window]
|
Figure 3. Dose-response curves for the active peptides.
Intracellular cAMP levels were measured in CHO/GLP-1R cells after
treatment with the indicated concentrations of GLP-1,
GLP-1 Gly8, and GLP-1
Aha8 for 30 min at 37 C. The data are normalized to maximum
values obtained in each experiment in the presence of
GLP-1 (10 nM). Bars represent
the mean ± SEM of three experiments preformed in
triplicate.
|
|
View larger version (16K):
[in this window]
[in a new window]
|
Figure 4. Displacement of
[125I]GLP-1 binding to CHO/GLP-1R cells with
the analogs of GLP-1.
[125I]GLP-1 binding to intact CHO/GLPR cells
was competed with various concentrations of the peptides shown. The
data are normalized to maximum values obtained in the presence of 10
nM of the respective peptides. The data
points represent the mean ± SEM of three
experiments preformed in triplicate. The IC50 value for
each GLP-1 analog is shown in Table 2 . Bo,
Maximum binding in the absence of cold peptide.
|
|
|
Results
|
|---|
In vitro static insulin secretion from RIN 1046-38 cells
The biological activity of the peptides was determined by
measuring the amount of insulin secreted into the medium of RIN 1046-38
cells over a 1-h period. The concentration at which maximum insulin
secretion was obtained with GLP-1 (10 nM) was
used to assess the potential of the peptides as insulin secretagogues.
Figure 1 shows the results of this
experiment. GLP-1 Aha8 was as
effective as GLP-1 in this assay, causing approximately a
3-fold increase in insulin release above that seen with glucose (5
mM) alone. Both GLP-1
Aha8 and GLP-1
Gly8 appeared to be as potent as
GLP-1 while inducing insulin secretion 1.2-fold above
basal at concentrations as low as 0.5 nM (data not shown).
Although the addition of one Aha moiety between positions 7 and 8 of
GLP-1 resulted in insulin secretion levels comparable to
those of GLP-1, the insertion of additional groups between
positions 8 and 9 abrogated the peptides’ biological potency.
View larger version (21K):
[in this window]
[in a new window]
|
Figure 1. Static in vitro insulin secretion.
RIN 1046-38 cells were incubated with glucose (5 mM) in the
absence or presence of GLP-1 (10 nM) or
GLP-1 analogs (10 nM) for 1 h at 37 C.
The amount of insulin secreted into the medium was then determined.
Data are the mean ± SEM of two or three experiments
performed in triplicate. **, P <
0.001; *, P < 0.05 (treated vs.
basal).
|
|
Replacing the L-amino acids with the
D-configuration reduced considerably the ability of the
compounds to induce insulin secretion. This was the case regardless of
the extent of substitution throughout the GLP-1
molecule.
In vitro intracellular cAMP production
Intracellular cAMP levels generated by the GLP-1
analogs were assessed initially at a peptide concentration of 10
nM (concentration at which maximum cAMP production is seen
with GLP-1). The peptides were incubated with the
CHO/GLP-1 R cells in the presence of IBMX for 30 min at 37
C. The results are shown in Fig. 2. In
agreement with the results from the in vitro insulin assay,
the D-amino acid substitutions throughout the
GLP-1 molecule resulted in only a small increase above
basal levels, i.e. those obtained with IBMX alone. Also,
GLP-1 (Aha9)4
and GLP-1
(Aha9)8 were inactive
compared with the insulinotropic compounds, GLP-1
Gly8 and GLP-1
Aha8. The induction of cAMP in response to
varying concentrations of GLP-1, GLP-1
Gly8, and GLP-1
Aha8 was measured (Fig. 3). Table 2 shows the
ED50 values of all three compounds.
GLP-1 Aha8 (0.5
nM) stimulated intracellular cAMP production to
4-fold above basal; however, it exhibited a higher
ED50 compared with GLP-1 and
GLP-1 Gly8.
View larger version (22K):
[in this window]
[in a new window]
|
Figure 2. Effect of GLP-1 analogs on the
production of intracellular cAMP. CHO/GLP-1R cells were incubated with
the indicated peptides (10 nM) for 30 min at 37 C, after
which they were lysed, and the lysates were processed for determination
of cAMP content. The data are normalized to maximum values obtained in
the presence of GLP-1 (10 nM). The
data points represent the mean of two or
three experiments. **, P < 0.001; *,
P < 0.05 (treated vs. basal).
|
|
Competitive binding of GLP-1 analogs to the
GLP-1 receptor
The potential of these GLP-1 analogs to displace
[125I]GLP-1 by binding
competitively to the human GLP-1 receptor was then
examined. CHO/GLP-1R cells were incubated with
125I-labeled GLP-1 in the absence
and presence of varying concentrations of the peptides (Fig. 4).
The IC50 values obtained for those compounds that
bound competitively to the GLP-1 receptor are shown in
Table 2. As shown previously, substitution of the glycine in the 8
position reduces the binding to the GLP-1R (5, 6). Insertion of the Aha moiety also resulted in a reduction in
binding to the receptor. With the increase in the length of the spacer
Aha groups in the 9 position, there was a dramatic decrease in affinity
for the GLP-1R. The lack of biological activity seen with
the D-amino acid-substituted compounds can be explained by
their markedly reduced ability to bind to the GLP-1R.
There was a progressive reduction in receptor recognition with
increasing D-amino acid substitution, such that compounds
GLP-1 D21 and GLP-1 All D did not displace
the labeled GLP-1.
Stability of GLP-1 Aha8 in vitro
The stability of GLP-1 Aha8 was
compared with that of GLP-1 in the presence of DPP IV and
human serum. Treatment of GLP-1 (2 µM) with
DPP IV (5 mU) for 10 min or with 100% serum for 2 h at 37 C
caused a considerable increase in the amount of N-terminal truncated
product (mol wt, 3089 g/mol) as measured by MALDI (Fig. 5, A–C). In contrast, GLP-1
Aha8 (2 µM) appeared resistant to
either treatment (Fig. 5, D–F).
View larger version (22K):
[in this window]
[in a new window]
|
Figure 5. Degradation of GLP-1 and
GLP-1 Aha8 by DPP IV and human serum. A–C,
MALDI mass spectroscopy of GLP-1: untreated (A), after
incubation with DPP IV for 10 min at 37 C (B), and after treatment with
human serum for 2 h at 37 C (C). D–F, MALDI mass spectroscopy of
GLP-1 Aha8: untreated (D), after incubation
with DPP IV at 37 C for 2 h (E), and after incubation with human
serum at 37 C for 2 h (F).
|
|
In vivo biological activity of GLP-1
Aha8
To verify that GLP-1 Aha8 had
biological activity in vivo, we administered the peptide sc
(24 nmol/kg) to fasted Zucker fatty (fa/fa) and Wistar rats.
Another group of Zucker rats received a similar dose of
GLP-1 Gly8. Blood glucose was then
monitored for the next 8 h. In Fig. 6 the results show that both compounds
rapidly lowered blood glucose. In Zucker rats the reduction in blood
glucose was more pronounced with GLP-1
Gly8 due to the fact that the fasting glucose
level was lower in that group but the slope and magnitude of the
decline were similar for both compounds. Insulin secretion was
attenuated in the GLP-1 Gly8 group
due to the drop in blood glucose into the hypoglycemic range, proving
the glucose dependency of insulinotropism with this class of compounds.
As the GLP-1 Aha8-treated Zucker
rats did not become hypoglycemic, the insulinotropic response did not
become abrogated, and the prolonged effect can be seen. In Wistar rats,
which are not hyperglycemic in the fasting state, insulin levels
increased rapidly with GLP-1 Aha8,
leading to hypoglycemia and, again, rapid attenuation of the
insulinotropic response.
View larger version (18K):
[in this window]
[in a new window]
|
Figure 6. The biological effects of GLP-1
Gly8 and GLP-1 Aha8. Blood glucose
and insulin levels were determined after sc injection of
GLP-1 Aha8 (24 nmol/kg) to Wistar and Zucker
fatty rats and of GLP-1 Gly8 (24 nmol/kg) to
Zucker rats only that had been fasted overnight. The results are the
mean ± SEM (n = 6/group).
|
|
|
Discussion
|
|---|
Here we have used three new strategies in an attempt to circumvent
cleavage of GLP-1 by DPP IV and preserve its
insulinotropic activity. These are 1) insertion of a single spacer
group between the 7 and 8 positions, 2) substitution of the naturally
occurring L-amino acid configuration with the corresponding
D-amino acid, and 3) insertion of multiple Aha groups in
between the 8 and 9 positions. Only the first strategy resulted in a
compound that proved to have a potency similar to that of
GLP-1 and GLP-1 Gly8.
The inclusion of a single Aha resulted in a compound that 1) binds to
the GLP-1 receptor, 2) stimulates cAMP production (at
concentrations of 0.5 nM and greater), 3) stimulates
insulin secretion in vitro, and 4) was effective in reducing
blood glucose levels in Zucker fatty rats for up to 8 h. In
contrast, the change in the amino acid stereochemistry resulted in a
series of biologically inactive compounds, as did the inclusion of
multiple spacer groups.
Previous modifications to the N-terminal end of the GLP-1
molecule have resulted in effective insulinotropic reagents that are
resistant to degradation by DPP IV (5, 6, 7). The insertion
of Aha in the position between the histidine and the alanine groups is
a novel strategy. The GLP-1 Aha8
analog has insulinotropic activity in vitro at 10
nM (maximum insulin secretory capacity for
GLP-1) similar to that seen with GLP-1 and
its DPP IV-resistant analog GLP-1
Gly8. As with all previous modifications at the 8
position, there is a reduction in affinity for the GLP-1
receptor, although to a lesser extent than is the case with
GLP-1 Gly8. Insertion of the single
Aha group resulted in a reduced capacity to generate intracellular cAMP
in CHO/GLP-1R cells relative to both GLP-1 and
GLP-1 Gly8. Although the
GLP-1 Aha8 compound binds more
avidly to the GLP-1 receptor, it is less potent in the
in vitro cAMP assay. Although binding is necessary for
ligand action, it does not reflect biological activity, as
demonstrated by the fact that cAMP response is actually less with
GLP-1 Aha8. Both GLP-1
Gly8 and GLP-1
Aha8 caused a significant decrease in blood
glucose levels by 30 min when administered sc to fasted Zucker fatty
rats.
Partial D-amino acid substitution has been used to improve
the stability of biologically active peptides (12) and
also as a structural probe (13). Siegel and colleagues
(14) have shown that substitution of the
D-histidine at position 7 results in a 9-fold reduction in
affinity of this analog for the GLP-1 receptor.
Substitution of D-alanine in the 8 position of
GLP-1 does not result in such a dramatic reduction in
receptor recognition of this compound and increases its stability
in vivo (14). All of our
D-amino acid compounds show a marked reduction in
binding affinity for the GLP-1 receptor. There is very
little change in the values for the IC50 between
the GLP-1 D3, GLP-1 D5, and
GLP-1 D8 compounds. Hence, the reduced binding affinities
of these compounds could be ascribed entirely to the change in
stereochemistry at the 7 position. Adelhorst and colleagues
(15) performed an alanine scan on the GLP-1
molecule and demonstrated that positions 7, 10, 12, 13, 15, 28, and 29
are critical for receptor binding. Substitution of the
D-amino acids up to positions 21 and 36 in the
GLP-1 molecule results in almost a complete loss of
receptor recognition, probably due to a distortion of the
stereochemical arrangement of the molecule as a whole. This marked
reduction in affinity for the GLP-1 receptor is reflected
in the virtual loss of biological activity. None of the
D-amino acid analogs showed any measurable effect
on intracellular cAMP formation in CHO/GLP-1R cells. Correspondingly,
there was very little stimulation of insulin secretion in the RIN
1046-38 cells treated with these peptides (10
nM).
The insertion of 4 and 8 Aha moieties in between positions 8 and 9 of
the GLP-1 sequence results in a reduction of biological
activity that is directly attributable to a reduced affinity for the
receptor and not to any antagonistic effect. However, the inclusion of
such long spacer arm in this region of the molecule does not completely
eliminate binding to the receptor. This concurs with the observations
made on the necessity of the histidine residue in position 7 for
receptor recognition vs. the mere reduction in affinity seen
with the modifications in the vicinity of the 8 position.
In summary we have shown that insertion of an Aha group after the 7
amino acid results in a compound with biological action of greater
duration than that of GLP-1 Gly8
in vivo. We have demonstrated that insertion of the Aha
group conferred protection against DPP IV, making this compound an
effective insulinotropic GLP-1 analog both in
vitro and in vivo. This strategy could be applied to
other peptides subject to degradation by DPP IV, e.g.
glucose-dependent insulinotropic polypeptide.
|
Acknowledgments
|
|---|
We thank Denis Muller, Diabetes Section, NIA, NIH, for
calculating the IC50 and
ED50 values. We thank Dr. Michael Baynham of the
Bioanalytical Chemistry Section, NIA, NIH, and Mr. Raynard Speiss
(Micromass, Cheshire, UK) for performing the MALDI
spectroscopy. Without the positive and on-going support of our
scientific director, Dr. D. L. Longo, these studies would not
have been carried out.
|
Footnotes
|
|---|
Abbreviations: Aha, 6-Aminohexanoic acid; CHO, Chinese
hamster ovary; DPP IV, dipeptidyl peptidase IV; GLP-1,
glucagon-like peptide-1; GLP-1R, glucagon-like peptide-1
receptor; IBMX, isobutylmethylxanthine; KRP, Krebs-Ringer
phosphate buffer; MALDI-TOF, matrix-assisted linear desorption
ionization-time of flight.
Received March 2, 2001.
Accepted for publication June 12, 2001.
|
References
|
|---|
-
Todd JF, Wilding JP, Edwards CM, Khan FA, Ghatei
MA, Bloom SR 1997 Glucagon-like peptide-1 (GLP-1): a trial of
treatment in non-insulin-dependent diabetes mellitus. Eur J Clin
Invest 27:533–536[CrossRef][Medline]
-
Orskov C, Rabenhoj L, Wettergren A, Kofod H, Holst
JJ 1994 Tissue and plasma concentrations of amidated and
glycine-extended glucagon-like peptide I in humans. Diabetes 43:535–539[Abstract]
-
Hansen L, Deacon CF, Orskov C, Holst JJ 1999 Glucagon-like peptide-1-(7–36)amide is transformed to glucagon-like
peptide-1-(9–36)amide by dipeptidyl peptidase IV in the capillaries
supplying the L cells of the porcine intestine. Endocrinology 140:5356–5363[Abstract/Free Full Text]
-
Elahi D, McAloon-Dyke M, Fukagawa NK, et al. 1994 The insulinotropic actions of glucose-dependent insulinotropic
polypeptide (GIP) glucagon-like peptide-1 (7–37) in normal and
diabetic subjects. Regul Pept 51:63–74[CrossRef][Medline]
-
Deacon CF, Knudsen LB, Madsen K, Wiberg FC, Jacobsen O,
Holst JJ 1998 Dipeptidyl peptidase IV resistant analogues of
glucagon-like peptide-1 which have extended metabolic stability and
improved biological activity. Diabetologia 41:271–278[CrossRef][Medline]
-
Burcelin R, Dolci W, Thorens B 1999 Long-lasting
antidiabetic effect of a dipeptidyl peptidase IV-resistant analog of
glucagon-like peptide-1. Metabolism 48:252–258[CrossRef][Medline]
-
O’Harte FP, Mooney MH, Lawlor A, Flatt PR 2000 N-Terminally modified glucagon-like peptide-1(7–36)amide exhibits
resistance to enzymatic degradation while maintaining its
antihyperglycaemic activity in vivo. Biochim Biophys Acta 1474:13–22[Medline]
-
Hupe-Sodmann K, McGregor GP, Bridenbaugh R, et al. 1995 Characterisation of the processing by human neutral endopeptidase
24.11 of GLP-1(7–36)amide and comparison of the substrate specificity
of the enzyme for other glucagon-like peptides. Regul Pept 58:149–156[CrossRef][Medline]
-
Montrose-Rafizadeh C, Egan JM, Roth J 1994 Incretin hormones regulate glucose dependent insulin secretion in RIN
1046-38 cells: mechanisms of action. Endocrinology 135:589–594[Abstract]
-
Montrose-Rafizadeh C, Yang H, Rodgers BD, Beday A,
Pritchette LA, Eng J 1997 High potency antagonists of the
pancreatic glucagon-like peptide-1 receptor. J Biol Chem 272:21201–21206[Abstract/Free Full Text]
-
Delean A, Munson PJ, Gaurdabasso V, Rodbard D 1988 A user’s guide to ALLFIT. Bethesda: NIH
-
Hong SY, Oh JE, Lee KH 1999 Effect of D-amino acid
substitution on the stability, the secondary structure, and the
activity of membrane-active peptide. Biochem Pharmacol 58:1775–1780[CrossRef][Medline]
-
Rothemund S, Beyermann M, Krause E, et al. 1995 Structure effects of double D-amino acid replacements: a nuclear
magnetic resonance and circular dichroism study using amphipathic model
helices. Biochemistry 1995 40:12954–12962
-
Siegel EG, Gallwitz B, Scharf G, et al. 1999 Biological activity of GLP-1-analogues with N-terminal modifications.
Regul Pept 79:93–102[CrossRef][Medline]
-
Adelhorst K, Hedegaard BB, Knudsen LB, Kirk O 1994 Structure-activity studies of glucagon-like peptide-1. J Biol Chem 269:6275–6278[Abstract/Free Full Text]
This article has been cited by other articles:
|
|
|
|
 
C M. B Edwards
GLP-1: target for a new class of antidiabetic agents?
J R Soc Med,
June 1, 2004;
97(6):
270 - 274.
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. E. Doyle and J. M. Egan
Pharmacological Agents That Directly Modulate Insulin Secretion
Pharmacol. Rev.,
March 1, 2003;
55(1):
105 - 131.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. E. Mayo, L. J. Miller, D. Bataille, S. Dalle, B. Goke, B. Thorens, and D. J. Drucker
International Union of Pharmacology. XXXV. The Glucagon Receptor Family
Pharmacol. Rev.,
March 1, 2003;
55(1):
167 - 194.
[Abstract]
[Full Text]
[PDF]
|
|
|
Top
Abstract
Introduction
