A Novel Glucagon Receptor Antagonist Inhibits
Glucagon-Mediated Biological Effects
Sajjad A. Qureshi,1 Mari Rios Candelore,1 Dan Xie,1 Xiaodong Yang,1 Laurie M. Tota,1
Victor D.-H. Ding,1 Zhihua Li,1 Alka Bansal,2 Corin Miller,3 Sheila M. Cohen,3 Guoqiang Jiang,1
Ed Brady,4 Richard Saperstein,4 Joseph L. Duffy,5 James R. Tata,5 Kevin T. Chapman,5
David E. Moller,1 and Bei B. Zhang1
Glucagon maintains glucose homeostasis during the
fasting state by promoting hepatic gluconeogenesis and
glycogenolysis. Hyperglucagonemia and/or an elevated
glucagon-to-insulin ratio have been reported in diabetic
patients and animals. Antagonizing the glucagon receptor is expected to result in reduced hepatic glucose
overproduction, leading to overall glycemic control.
Here we report the discovery and characterization of
compound 1 (Cpd 1), a compound that inhibits binding
of 125I-labeled glucagon to the human glucagon receptor
with a half-maximal inhibitory concentration value of
181 ⴞ 10 nmol/l. In CHO cells overexpressing the human
glucagon receptor, Cpd 1 increased the half-maximal
effect for glucagon stimulation of adenylyl cyclase with
a KDB of 81 ⴞ 11 nmol/l. In addition, Cpd 1 blocked
glucagon-mediated glycogenolysis in primary human
hepatocytes. In contrast, a structurally related analog
(Cpd 2) was not effective in blocking glucagon-mediated
biological effects. Real-time measurement of glycogen
synthesis and breakdown in perfused mouse liver
showed that Cpd 1 is capable of blocking glucagoninduced glycogenolysis in a dosage-dependent manner.
Finally, when dosed in humanized mice, Cpd 1 blocked
the rise of glucose levels observed after intraperitoneal
administration of exogenous glucagon. Taken together,
these data suggest that Cpd 1 is a potent glucagon
receptor antagonist that has the capability to block the
effects of glucagon in vivo. Diabetes 53:3267–3273, 2004
From the 1Department of Metabolic Disorder and Molecular Endocrinology,
Merck Research Laboratories, Rahway, New Jersey; the 2Department of
Human-Animal Infectious Disease Research, Merck Research Laboratories,
Rahway, New Jersey; the 3Department of Image Research, Merck Research
Laboratories, Rahway, New Jersey; the 4Department of Pharmacology, Merck
Research Laboratories, Rahway, New Jersey; and the 5Department of Medicinal Chemistry, Merck Research Laboratories, Rahway, New Jersey.
Address correspondence and reprint requests to Dr. Sajjad A. Qureshi,
RY80N-A62, Merck Research Laboratories, P.O. Box 2000, Rahway, NJ 07065.
E-mail: [email protected].
Received for publication 7 June 2004 and accepted in revised form 27
August 2004.
S.A.Q. and M.R.C. contributed equally to this article.
GCGR, glucagon receptor; GIP, glucose-dependent insulinotropic peptide;
GPCR, G-protein– coupled receptor; HCM, hepatocyte culture medium;
hGCGR, human GCGR; hGIPR, human glucose insulinotropic peptide receptor; HGP, hepatic glucose production; IMDM, Iscove’s modified Dulbecco’s
medium; NMR, nuclear magnetic resonance.
© 2004 by the American Diabetes Association.
DIABETES, VOL. 53, DECEMBER 2004
G
lucagon is a 29⫺amino acid polypeptide produced in the pancreatic ␣-cells and secreted in
response to falling glucose levels during the
fasting period (1). Glucagon increases glucose
production by promoting glycogenolysis and gluconeogenesis in the liver and attenuation of the ability of insulin to
inhibit these processes (2). The combined action of glucagon and insulin is responsible for maintaining whole-body
glucose homeostasis (3). Both increased glucagon secretion during the fasting state and the lack of insulinmediated suppression of glucagon production in
postprandial state contribute to the elevated glucagon
levels associated with the hyperglycemia observed in the
diabetic state (4,5). Therefore, reducing circulating glucagon levels and inhibiting glucagon-mediated biological
effects in target tissues have long been considered as
means of reducing hyperglycemia in diabetes. Studies
using potent peptide antagonists have demonstrated significant blood glucose⫺lowering effects in diabetic animal
models (6,7). Furthermore, it has been demonstrated that
immunoneutralization of glucagon in diabetic animals
effectively diminishes glucagon-stimulated hyperglycemia
(8 –10). Although these reagents have shown promising
results in animal models, their development for use in
humans has not progressed because of limitations imposed by the delivery methods necessary to achieve
significant levels of exposure for the peptide agents.
The glucagon receptor (GCGR) is a member of the
family B of the seven transmembrane G-protein– coupled
receptor (GPCR) superfamily (11). Other closely related
members of the family include the receptors for glucagonlike peptide 1 and glucose-dependent insulinotropic peptide (GIP). Glucagon signals by binding to the receptor,
which leads to activation of adenylyl cyclase and an
increase in intracellular cAMP levels (12). In addition, the
GCGR also couples to an intracellular Ca2⫹-mediated
pathway (13). Activation of the GCGR results in increased
glycogenolysis and gluconeogenesis, which are responsible for increased hepatic glucose output (14,15).
Given the key role of glucagon in elevating glycemia and
owing to the success of finding small-molecule inhibitors
for many receptors in the GPCR family (16,17), the GCGR
is a clear target for the development of small-molecule
antagonists. A number of antagonists with varying degree
of potency and structures have been reported in recent
3267
GLUCAGON RECEPTOR ANTAGONIST
years (rev. in 18). Among them, one compound (Bay-279955) advanced to evaluation in humans in early clinical
trials and was shown to block glucagon-stimulated glucose
production in healthy volunteers (19). Although Bay-279955 has apparently not been studied in diabetic patients,
current results with the compound do provide further
validation for the notion that antagonism of the GCGR may
be a viable approach to regulating glycemia in humans.
Thus, there is now a compelling need to identify newer
and better optimized GCGR antagonists that could be
considered as alternative clinical development candidates
for the treatment of type 2 diabetes.
In our study, we identified compounds in a new chemical class that are potent in blocking glucagon-dependent
activation of the human GCGR in human hepatocytes and
in a mouse model expressing the human GCGR. Here we
describe the characterization of a representative compound from this novel class of glucagon antagonists.
RESEARCH DESIGN AND METHODS
Chinese hamster ovary (CHO) cells expressing the cloned human GCGR
(CHO-hGCGR) have been previously described (20,21). The CHO-hGIPR cell
line was produced by transfecting human glucose insulinotropic peptide
receptor (hGIPR) cDNA (kindly provided by T. Usdin, National Institutes of
Health, Bethesda, MD) into CHO cells. All cell lines were maintained in
Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% FBS, 1
mmol/l L-glutamine, penicillin-streptomycin (100 units/ml), and G418 (500
␮g/ml) (GIBCO/Invitrogen Life Sciences). Primary human hepatocytes were
obtained from In Vitro Technologies (Baltimore, MD) and maintained in
hepatocyte culture medium (HCM) (Clonetics, PA). Compounds (Cpds) 1 and
2 were initially procured from Olivia Scientific (Princeton, NJ) and later
synthesized in house by standard chemical synthesis (J.L.D., unpublished
observations). Stock solutions of compounds were prepared in 100% DMSO
and diluted in the appropriate buffer just before being used. All other materials
were from commercial sources.
Male mice used in this study originated from a humanized GCGR mouse
line generated by replacing the mouse GCGR gene with that of the hGCGR
gene, as previously described (22). Animals were housed in a standard rodent
facility with free access to standard rodent diet and water following standard
institutional guidelines for animal care. All animal care and procedures were
performed in accordance with institutional guidelines.
Glucagon receptorⴚbinding assay. To determine the binding affinity of
selected compounds, 2 mg of cell membranes from the CHO-hGCGR cells
were incubated with 50 pmol/l 125I-labeled glucagon (Du Pont-NEN) in a buffer
containing 50 mmol/l Tris HCl (pH 7.5), 5 mmol/l MgCl2, 2 mmol/l EDTA, 12%
glycerol, and 200 mg wheat germ agglutinin-coated polyvinyltoluene scintillation proximity assay beads (Amersham) with or without increasing concentrations of the compounds. Nonspecific binding was determined in the same
buffer in the presence of 1 ␮mol/l unlabeled glucagon. After a 4- to 12-h
incubation at room temperature, the radioactivity bound to the cell membranes was determined in a scintillation counter (Microbeta; PerkinElmer Life
Sciences, Boston, MA). The half-maximal inhibitory concentration value (IC50)
for native glucagon was 2.8 ⫾ 0.4 nmol/l (n ⫽ 5).
Adenylyl cyclase assay. cAMP levels in CHO-hGCGRs or CHO-hGIPRs were
determined with the aid of an adenylyl cyclase assay kit (SMP-004B,
PerkinElmer Life Sciences), per the manufacturer’s instructions. To assess
antagonist activity, cells were incubated with Cpd 1 for 30 min and then
stimulated with glucagon (0 –500 nmol/l), forskolin (1 ␮mol/l), or GIP (250
pmol/l) for 30 min. The cell stimulation was stopped by adding an equal
amount of a detection buffer containing cell lysis agent and 125I-labeled cAMP
tracer. The amount of 125I-cAMP bound to the plate was determined using a
liquid scintillation counter (Microbeta; PerkinElmer Life Sciences) and used
to quantitate the amount of cAMP present in each sample. The half-maximal
effect (EC50) for glucagon in CHO-hGCGR cells was 0.22 ⫾ 0.2 nmol/l (n ⫽ 5).
The Kdb and pA2 values were determined by the method of Arunlakshana and
Schild (23).
␤-Lactamase reporter gene assay. A glucagon-responsive ␤-lactamase
reporter gene cell line was established by transfecting the hGCGR into a CHO
cell line containing a ␤-lactamase reporter gene under the control of a
cAMP-responsive promoter (24); cells that expressed the ␤-lactamase gene in
response to stimulation with 100 nmol/l glucagon were selected as previously
described (24; S.A.Q. and B.B.Z., unpublished observations). A cell line
3268
expressing the hGIPR was prepared using the same protocol, except that
GIP-responsive cells were selected. To study changes in cAMP-dependent
transcription in these cells, 5,000 cells per well were plated onto 96-well,
clear-bottomed plates and allowed to adhere to the plate for ⬃18 –24 h.
Subsequently, the medium was removed and each well was washed with
IMDM containing 0.1% FBS and incubated with one of the compounds or 1%
DMSO (as a control) in the same medium for 30 min. Cells were stimulated
with 250 pmol/l glucagon, 1 ␮mol/l forskolin, or 100 pmol/l GIP (for cells
expressing the hGIPR) for 4 h and then loaded with CCF2/AM (a ␤-lactamase
substrate dye) to detect ␤-lactamase activity, as previously described (24).
Glycogen synthesis assay. Fresh human primary hepatocytes were plated
(1.7 ⫻ 106/well) onto collagen-coated, six-well plates in HCM BulletKit
medium (Clonetics); a day later, they were serum starved overnight by
switching to HCM with low-glucose (1 g/l) medium. Glycogen synthesis and
labeling were initiated by adding 200 nmol/l insulin and D-[U-14C]glucose (2
␮Ci/ml; specific activity 0.36 ␮Ci/␮mol; PerkinElmer Life Sciences) in the
same medium. After being incubated for 3 h at 37°C, the cells were treated
with glucagon (2 nmol/l) in presence of DMSO alone or one of the compounds
for an additional 60 min. Cell glycogen contents were measured as previously
described (25).
Inhibition of glycogen breakdown in perfused mouse liver. Glycogen
levels in perfused mouse liver were determined as previously described
(26,27). Briefly, [2-13C]pyruvate was incorporated into endogenous glycogen
and 13C nuclear magnetic resonance (NMR) spectroscopy was used to monitor
the synthesis and breakdown of [13C]glycogen in real time in perfused livers
from the hGCGR mice (26). Cpd 1 or 2 diluted from a stock in 100% DMSO was
added to the perfusate to yield the desired concentration of compound. In a
similar fashion, human glucagon prepared in normal saline was added to the
perfusate at the indicated time. The amount of glycogen at a given time was
estimated from the area of the C1 resonance of the glucosyl unit of glycogen.
Absolute areas were normalized by the area of the glycogen C1 resonance at
t ⫽ 0 (glucagon administration).
Glucagon challenge assay in mice with the human glucagon receptor.
Male mice with the hGCGR (age 10 –11 weeks) (22) were used in these
studies. These mice had free access to standard rodent diet and water before
being dosed with a compound; food was withdrawn right before the dosing
with vehicle or compound. Animals were given either vehicle (60% polyethylene glycol, 20% ethanol, and 20% saline) or compound suspension in the
vehicle via an intraperitoneal injection at the indicated dosage. At 1 h after the
first intraperitoneal dose, a bolus dose of 15 ␮g/kg glucagon (Eli Lilly) in
normal saline containing 0.5% BSA or normal saline plus 0.5% BSA was
administered via a second intraperitoneal injection. Blood glucose levels were
monitored using a One Touch Basic Glucometer (Lifescan) before vehicle or
compound was injected (t ⫽ ⫺60) and before the saline or glucagon
intraperitoneal injection (t ⫽ 0) and at 7.5, 15, 30, and 45 min after glucagon
challenge.
Statistical analysis. IC50 and EC50 values were obtained using the nonlinear
regression analysis (curve fit) assuming single site binding. P values were
calculated using the unpaired Student’s t test. All data were analyzed with the
aid of GraphPad Prism software (GraphPad Software, San Diego, CA).
RESULTS
Identification of a selective glucagon receptor antagonist. To identify novel GCGR antagonists, we screened
chemical libraries for compounds that block 125I-glucagon
binding to membranes prepared from CHO-hGCGR cells.
From our search, we identified N-[3-cyano-6-(1, 1-dimethylpropyl)-4, 5, 6, 7-tetrahydro-1-benzothien-2-yl]-2-ethylbutanamide as a potent GCGR antagonist (Cpd 1) (Fig. 1A).
Cpd 1 inhibited the binding of 125I-glucagon to the membrane with an IC50 of 181 ⫾ 10 nmol/l (n ⫽ 3). Cpd 1
specifically interacts with the hGCGR, as it did not inhibit
the binding of cognate ligands to a number of other
GPCRs, such as the receptors for GIP and neuropeptide Y
(data not shown). In contrast, Cpd 2, a structurally related
analog, was a poor inhibitor (20 ⫾ 1.5% inhibition at 10
␮mol/l) of glucagon binding (Fig. 1B).
Binding of glucagon to its receptor resulted in activation
of adenylyl cyclase and subsequently cAMP production
(12), which is responsible for many of glucagon’s actions,
including activation of glycogen phosphorylase and inhiDIABETES, VOL. 53, DECEMBER 2004
S.A. QURESHI AND ASSOCIATES
FIG. 2. Attenuation of glucagon-stimulated adenylyl cyclase activation
by Cpd 1 in CHO-hGCGR cells. Data show glucagon dosage response in
CHO-hGCGR cells preincubated in the presence of DMSO or Cpd 1 at
the indicated dosages for 30 min. Data are normalized to the maximum
glucagon response observed in cells treated with 500 nmol/l glucagon in
the presence of DMSO alone (assigned as 100%) and are means of three
replicates.
FIG. 1. Characteristics of novel glucagon receptor antagonists.
A: Structure of Cpds 1 and 2. B: Inhibition of the binding of [125I]glucagon to the hGCGR by Cpds 1 and 2 in the receptor-binding assay
described in RESEARCH DESIGN AND METHODS. Data are means of three
replicates
bition of glycogen synthase. Increasing the concentration
of Cpd 1 increased the EC50 for glucagon-dependent
adenylyl cyclase activation in CHO-hGCGR cells without
affecting the maximal glucagon response (Fig. 2). This
result suggested that Cpd 1 is a competitive antagonist, a
conclusion further supported by the Schild transformation
(23) of data shown in Fig. 2 that yields a straight line with
a slope not significantly different from unity. Moreover, the
affinity of Cpd 1 for the GCGR, as measured by the
concentration of the antagonist that shifted the agonist
dosage response by twofold (KDB), was estimated as 81 ⫾
11 nmol/l (n ⫽ 3) and pA2 (the negative logarithm of the
inhibitor concentration that reduces the agonist response
to 1 unit of glucagon to the response obtained with 0.5
units of glucagon) as 7.1 ⫾ 0.06 (23). The effect of Cpd 1
was specific to the GCGR, as it had no effect on either
forskolin-dependent cAMP production in the same cells or
GIP-dependent cAMP production in cells expressing
hGIPRs (data not shown). In contrast, Cpd 2 showed a
⬍10% inhibition of glucagon, forskolin, and GIP-induced
cAMP production.
Activation of the GCGR leads to increased gene expression of enzymes involved in gluconeogenesis, such as
glucose 6-phosphatase and PEPCK (14,15). To establish
that Cpd 1 is able to suppress glucagon-induced gene
DIABETES, VOL. 53, DECEMBER 2004
expression, we examined the agent’s effect on the induction of a cAMP-responsive ␤-lactamase reporter gene in
CHO cells. The ␤-lactamase reporter gene cell line was
prepared by introducing the hGCGR into cells expressing
a ␤-lactamase reporter gene under the control of a cAMPresponsive promoter element (24). Glucagon increased
␤-lactamase activity in these cells with an EC50 of 250
pmol/l. When tested in this cell line, Cpd 1 inhibited the
glucagon-stimulated rise in the ␤-lactamase activity with
an IC50 of 1,569 ⫾ 374 nmol/l (n ⫽ 4), whereas Cpd 2 had
no effect in this assay. In addition, Cpd 1 had no effect on
the forskolin-stimulated rise in ␤-lactamase activity in
these cells or on GIP activity in the ␤-lactamase cell line
expressing hGIPR.
Taken together, these data demonstrate that Cpd 1 is a
selective and competitive GCGR antagonist.
Inhibition of glycogenolysis in human hepatocytes.
The binding of glucagon to its receptor results in increased
glycogenolysis that is believed to be responsible, at least in
part, for hepatic glucose production (HGP) during fasting
periods. To examine if Cpd 1 would block this effect, we
first induced glycogen synthesis in human primary hepatocytes with 200 nmol/l insulin in the presence of [14C]glucose to increase the pool of labeled glycogen. Incubation
of human hepatocytes under these conditions resulted in a
three- to fivefold increase in the amount of radiolabeled
glycogen in these cells (Fig. 3; compare the basal and
insulin-stimulated responses). As expected, treatment of
insulin-stimulated cells with glucagon reduced the [14C]
glycogen contents to basal levels. However, the addition of
Cpd 1 at 10 or 30 ␮mol/l along with glucagon significantly
attenuated the glucagon-induced glycogenolysis in this
setting. Once again, Cpd 2 at the same dosages was
ineffective in blocking the glucagon-induced glycogenolysis in these cells. These data demonstrate the ability of
Cpd 1 to block the glucagon-dependent glycogen breakdown in human hepatocytes, a target cell population for
the action of glucagon in vivo.
Inhibition of glycogenolysis in perfused liver. To
demonstrate that the inhibitory effect of Cpd 1 on the
3269
GLUCAGON RECEPTOR ANTAGONIST
FIG. 3. Inhibition of glucagon-induced glycogenolysis in human
primary hepatocytes. Primary human hepatocytes were incubated with [14C]glucose for 3 h without (basal) and with 200
nmol/l insulin (Ins) to facilitate labeling of glycogen. After the
labeling period, the samples were treated with 2 nmol/l glucagon (GCG) with or without Cpd 1 or 2 at the indicated dosages
for 60 min. Glycogen levels in each sample were then measured
as described in RESEARCH DESIGN AND METHODS. Data are means ⴞ
SE of three replicates and are taken from a representative
experiment. Qualitatively similar data were obtained in at least
three independent experiments; however, due to significant
variance in the response to insulin in various human hepatocytes preparations, pooling of the data has been avoided. P
values were calculated using the unpaired Student’s t test.
glycogenolysis observed in vitro in human hepatocytes
will also result in the inhibition of glucagon-dependent
glycogenolysis in vivo, the activity of the Cpd 1 was
examined by 13C NMR in intact perfused liver obtained
from humanized mice (22) in an ex vivo setting. In this
setting, the intact liver is maintained in an isotonic environment to measure various metabolites in real time at or
near-physiological conditions (26). To assess the effect of
Cpd 1 under these conditions, we first labeled endogenous
glycogen with the aid of [2-13C]pyruvate. Administration of
[2-13C]pyruvate (6.7 mmol/l) over the entire duration of
experiment (⬃150 min) resulted in a steady increase in the
amount of glycogen, as indicated by changes in the levels
of [13C]glycogen in these livers (Fig. 4). Furthermore, this
rise in glycogen synthesis was not affected by the introduction of Cpd 1, Cpd 2, or DMSO into the perfusate ⬃40
min after the start of labeling. Exposure of these livers to
glucagon (50 pmol/l) resulted in a rapid degradation of
glycogen in the DMSO-treated livers only. However, in Cpd
1⫺treated livers, a significant, dosage-dependent reduction in glucagon-induced glycogenolysis was observed
(Fig. 4). As observed earlier, Cpd 2, the inactive analog,
had no effect in this experimental setting. In addition,
vehicle-treated livers continued to synthesize glycogen at a
FIG. 4. Inhibition of glucagon-induced glycogenolysis in mouse liver. An intact liver from a male hGCGR mouse was obtained and quickly perfused
with a Krebs bicarbonate buffer containing 2.5% BSA at 37°C. Endogenous glycogen was labeled with the constant administration of
[2-13C]pyruvate in the same buffer that started at t ⴝ ⴚ90 min and continued throughout the experiment. At t ⴝ ⬃ⴚ25 min, Cpd 1 or 2 (at the
indicated dosages) or DMSO was added to the perfusate. At t ⴝ 0, glucagon (GCG) or saline was introduced into the perfusate. The glycogen levels
during the entire experiment were determined via 13C NMR spectra and are means ⴞ SE of three independent determinations.
3270
DIABETES, VOL. 53, DECEMBER 2004
S.A. QURESHI AND ASSOCIATES
FIG. 5. Inhibition of glucagon activity in hGCGR mice. hGCGR mice
were dosed with vehicle (Veh) or Cpd 1 at 15 and 50 mg/kg (mpk) in the
same vehicle via an intraperitoneal injection 1 h before glucagon
challenge. A bolus dose of glucagon (15 ␮g/kg) in normal saline (GCG)
or normal saline alone (saline) was administrated via a second intraperitoneal injection at t ⴝ 0. Glucose measurements were taken at the
indicated times after the glucagon challenge. Data are means ⴞ SE (n ⴝ
8 –10 animals per group).
rate similar to their previous rate. These data show that
Cpd 1 has the ability to block glucagon-induced glycogenolysis under near-physiological conditions in the target
organ of interest.
Inhibition of glucagon-induced glucose elevation in a
hGCGR mouse model. Administration of glucagon in
animal models as well as in humans results in a rapid rise
in plasma glucose levels due to glycogen breakdown
(7,19). We used this paradigm to evaluate the potential of
our compound to inhibit glucagon activity in vivo using the
hGCGR mouse model (22). To test the antagonistic potential of the compound, we first treated these animals with
Cpd 1 or vehicle via an intraperitoneal injection and then
added a glucagon challenge 1 h later. As shown in Fig. 5, a
single intraperitoneal injection of 15 ␮g/kg glucagon resulted in a rapid rise in blood glucose levels in vehicletreated animals, whereas the administration of a similar
volume of saline produced minimal change in blood glucose levels in these animals (compare vehicle/saline with
vehicle/GCG in Fig. 5). However, in the animals that had
been treated with Cpd 1 at 50 mg/kg (but not at 15 mg/kg)
before the glucagon challenge, the effect of glucagon was
significantly blunted. These data show that Cpd 1 is
capable of blocking the effect of glucagon in vivo, and thus
should affect the regulation of glucose homeostasis in the
diabetic state.
DISCUSSION
Blood glucose levels are maintained through the balance
between glucose uptake by the peripheral tissues and
glucose production and secretion by the liver. The liver
plays a central role in maintaining blood glucose levels by
acting as a storage depot during periods of dietary carbohydrate excess and as a glucose source when carbohydrate supply is scarce. Two pathways contribute to HGP:
the breakdown of glycogen (or glycogenolysis) and gluconeogenesis from precursors such as lactate, gluconeogenic amino acids, or glycerol. This homeostatic system is
under the tight control of hormones, with insulin playing a
major role in suppressing HGP under fed conditions and
DIABETES, VOL. 53, DECEMBER 2004
glucagon and corticosteroids promoting HGP under fasting conditions. An inappropriately high rate of HGP is the
predominant cause of fasting hyperglycemia and a major
contributor to the postprandial hyperglycemia characteristic of type 2 diabetes (28,29). In addition, increased
circulating glucagon levels are also linked to elevated HGP
in type 2 diabetes (3,30).
Glucagon and the GCGR represent potential targets for
the treatment of diabetes (31,32). Over the last two decades, encouraging progress has been made in attempts to
normalize hyperglycemia by antagonizing glucagon signaling using glucagon-neutralizing antibodies, peptide glucagon analogs that function as antagonists, and nonpeptide
small-molecule GCGR antagonists. High-affinity glucagonneutralizing antibodies can effectively reduce free glucagon and lead to reduced glycemia in animal models (9 –10).
Extensive efforts have been made to generate linear and
cyclic glucagon analog peptides. Compared with native
glucagon, some of these peptide analogs have shown
distinct properties in terms of their ability to bind to the
GCGR and affect glucagon-stimulated cAMP production.
They act as pure agonists, partial agonists/antagonists, or
pure antagonists of the GCGR (33–35). It was first reported
that [1-Na-trinitrophenylhistidine, 12-homoarginine]-glucagon, a potent antagonistic glucagon analog, significantly
decreased hyperglycemia in streptozotocin-induced diabetic rats in vivo (6). Other potent glucagon analogs have
also demonstrated efficacy in animal models of diabetes
(7).
The discovery and development of nonpeptidyl GCGR
antagonists of diverse structures have been reported over
recent years (rev. in 18). The published antagonists appear
to act via distinct receptor mechanisms. Skyrin, one of the
earlier antagonists, appeared to functionally inhibit glucagon-stimulated cAMP production and glycogenolysis without affecting glucagon binding (36). Other antagonists
inhibit both the binding and the function of glucagon in
competitive or noncompetitive fashion. Most recently,
Bay-27-9955, an orally absorbed and potent GCGR antagonist, has been shown to block glucagon-induced elevation of blood glucose in human (19).
Given the potential importance of this new therapeutic
approach and the apparent lack of progress made with
existing compounds, we sought to discover and characterize a new, and hopefully improved, class of GCGR antagonists. In this study, we showed that a new compound
specifically and competitively inhibits glucagon binding
and blocks glucagon-stimulated glycogenolysis in human
hepatocytes.
Glucagon receptor antagonists have been shown to
exhibit species specificity, as some antagonists are more
potent toward the human than the murine GCGR (20) and
vice versa. Therefore, the demonstration of efficacy for
novel structural classes may be challenging, as compounds are prioritized based on their activities using in
vitro assays in human receptors, but must be evaluated in
nonhuman animal models before their introduction in
humans. In this regard, a mouse model expressing the
hGCGR generated with a direct replacement vector is
highly valuable in evaluating the in vivo efficacy of GCGR
antagonists (22). In this study with humanized mice, we
characterized Cpd 1 using primary hepatocytes (F. Liu and
3271
GLUCAGON RECEPTOR ANTAGONIST
G.J., unpublished observations), perfused liver, and in vivo
testing. This study design provided a unique paradigm for
evaluating the potency of the antagonist compounds on
the hGCGR in a preclinical setting and thus should afford
better correlation with the potency of antagonists in future
studies in humans.
Because liver is the primary target tissue for glucagon
action, we evaluated the effect of our antagonist lead
compound in perfused liver using NMR spectroscopy
(26,27). This method enables a real-time assessment of
glycogen synthesis and glucagon-stimulated glycogenolysis in the intact organ under conditions that approximate
real physiology. Phosphorus spectra were taken in the
beginning and at the end of the perfusion studies to ensure
the presence of consistent levels of ATP, which was used
as an indicator that the perfused livers were in a healthy
state. In this study, we demonstrated that Cpd 1 was able
to attenuate glucagon-induced glycogen breakdown in a
dosage-dependent manner. These results were complementary to similar findings using human primary hepatocytes. Furthermore, we used the glucagon challenge
method to determine the in vivo activity of GCGR antagonists. This paradigm is analogous to the one used in the
reported human study of Bay-27-9955 and provides an
efficient means to evaluate antagonist compounds in an
animal model.
Hypoglycemia is a theoretical mechanism-based safety
concern for GCGR antagonists. However, in ob/ob mice
treated with antiglucagon antibodies for 1 week until
undetectable plasma glucagon levels were reached, significant reductions in elevated glucose and triglyceride levels
were observed without hypoglycemia (9). Furthermore,
GCGR knockout mice are not overtly hypoglycemic
(37,38). Similarly, GCGR antisense oligonucleotides reversed hyperglycemia in diabetic rodent models without
causing hypoglycemia (39,40). Of note, human glucose
counterregulation involves several pathways, mediated
not only by glucagon but also by catecholamines, corticosteroids, and growth hormone. Blocking the glucagon axis
should not interfere with these other counterregulatory
responses. Thus, severe hypoglycemia is not expected
with glucagon antagonists.
In summary, Cpd 1 represents a novel class of compound that functions to block glucagon binding and antagonize biological responses elicited by glucagon in human
hepatocytes. It is important to note that Cpd 1 also
blocked the effects of glucagon on perfused whole liver
from hGCGR mice and diminished glucagon-stimulated
glucose elevations in the mouse model. These results
indicate that additional optimized small-molecule GCGR
antagonists can be derived and suggest that efficient
blockade of glucagon action is an effective means to
control HGP and should lead to efficacy in lowering fasting
and postprandial hyperglycemia in type 2 diabetes.
ACKNOWLEDGMENTS
We are thankful to G. Chicchi for reagents and helpful
discussions for the studies. We thank K. Sullivan and L.
Shiao for providing the hGCGR mouse model. We are also
thankful to Emma Parmee for comments on the manuscript and D. Ashnault-Biazzo, J. Inglese, P. Fischer, G.
Koch, P. Kunapuli, F. Liu, D. Szalkowski, M. Wright, and Q.
3272
Dallas-Yang for technical assistance and useful discussions.
REFERENCES
1. Burcelin R, Katz EB, Charron MJ: Molecular and cellular aspects of the
glucagon receptor: role in diabetes and metabolism. Diabetes Metab
22:373–396, 1996
2. Consoli A: Role of liver in pathophysiology of NIDDM. Diabetes Care
15:430 – 441, 1992
3. Unger RH: Role of glucagon in the pathogenesis of diabetes: the status of
the controversy. Metabolism 27:1691–1709, 1978
4. Muller WA, Faloona GR, Aguilar-Parada E, Unger RH: Abnormal alpha-cell
function in diabetes: response to carbohydrate and protein ingestion.
N Engl J Med 283:109 –115, 1970
5. Reaven GM, Chen YD, Golay A, Swislocki AL, Jaspan JB: Documentation
of hyperglucagonemia throughout the day in nonobese and obese patients
with noninsulin-dependent diabetes mellitus. J Clin Endocrinol Metab
64:106 –110, 1987
6. Johnson DG, Goebel CU, Hruby VJ, Bregman MD, Trivedi D: Hyperglycemia of diabetic rats decreased by a glucagon receptor antagonist. Science
215:1115–1116, 1982
7. Unson CG, Gurzenda EM, Merrifield RB: Biological activities of desHis1[Glu9]glucagon amide, a glucagon antagonist. Peptides 10:1171–1177,
1989
8. Brand CL, Jorgensen PN, Knigge U, Warberg J, Svendsen I, Kristensen JS,
Holst JJ: Role of glucagon in maintenance of euglycemia in fed and fasted
rats. Am J Physiol 269:E469⫺E477, 1995
9. Brand CL, Rolin B, Jorgensen PN, Svendsen I, Kristensen JS, Holst JJ:
Immunoneutralization of endogenous glucagon with monoclonal glucagon
antibody normalizes hyperglycaemia in moderately streptozotocin-diabetic
rats. Diabetologia 37:985–993, 1994
10. Brand CL, Jorgensen PN, Svendsen I, Holst JJ: Evidence for a major role
for glucagon in regulation of plasma glucose in conscious, nondiabetic,
and alloxan-induced diabetic rabbits. Diabetes 45:1076 –1083, 1996
11. Pierce KL, Premont RT, Lefkowitz RJ: Seven-transmembrane receptors.
Nat Rev Mol Cell Biol 3:639 – 650, 2002
12. Lok S, Kuijper JL, Jelinek LJ, Kramer JM, Whitmore TE, Sprecher CA,
Mathewes S, Grant FJ, Biggs SH, Rosenberg GB: The human glucagon
receptor encoding gene: structure, cDNA sequence and chromosomal
localization. Gene 140:203–209, 1994
13. Hansen LH, Gromada J, Bouchelouche P, Whitmore T, Jelinek L, Kindsvogel W, Nishimura E: Glucagon-mediated Ca2⫹ signaling in BHK cells
expressing cloned human glucagon receptors. Am J Physiol 274:
C1552⫺C1562, 1998
14. Beale E, Andreone T, Koch S, Granner M, Granner D: Insulin and glucagon
regulate cytosolic phosphoenolpyruvate carboxykinase (GTP) mRNA in
rat liver. Diabetes 33:328 –332, 1984
15. Yoon JC, Puigserver P, Chen G, Donovan J, Wu Z, Rhee J, Adelmant G,
Stafford J, Kahn CR, Granner DK, Newgard CB, Spiegelman BM: Control of
hepatic gluconeogenesis through the transcriptional coactivator PGC-1.
Nature 413:131–138, 2001
16. Drews J: Drug discovery: a historical perspective. Science 287:1960 –1964,
2000
17. Davey J: G-protein-coupled receptors: new approaches to maximise the
impact of GPCRs in drug discovery. Expert Opin Ther Targets 8:165–170,
2004
18. Ling A: Small-molecule glucagon receptor antagonists. Drugs of the Future
27:987–993, 2002
19. Petersen KF, Sullivan JT: Effects of a novel glucagon receptor antagonist
(Bay 27-9955) on glucagon-stimulated glucose production in humans.
Diabetologia 44:2018 –2024, 2001
20. Cascieri MA, Koch GE, Ber E, Sadowski SJ, Louizides D, de Laszlo SE,
Hacker C, Hagmann WK, MacCoss M, Chicchi GG, Vicario PP: Characterization of a novel, non-peptidyl antagonist of the human glucagon receptor.
J Biol Chem 274:8694 – 8697, 1999
21. Chicchi GG, Graziano MP, Koch G, Hey P, Sullivan K, Vicario PP, Cascieri
MA: Alterations in receptor activation and divalent cation activation of
agonist binding by deletion of intracellular domains of the glucagon
receptor. J Biol Chem 272:7765–7769, 1997
22. Shiao LL, Cascieri MA, Trumbauer M, Chen H, Sullivan KA: Generation of
mice expressing the human glucagon receptor with a direct replacement
vector. Transgenic Res 8:295–302, 1999
23. Arunlakshana O, Schild HO: Some quantitative uses of drug antagonists.
Br J Pharmacol 14:48 –58, 1959
24. Kunapuli P, Ransom R, Murphy KL, Pettibone D, Kerby J, Grimwood S,
DIABETES, VOL. 53, DECEMBER 2004
S.A. QURESHI AND ASSOCIATES
Zuck P, Hodder P, Lacson R, Hoffman I, Inglese J, Strulovici B: Development of an intact cell reporter gene beta-lactamase assay for G proteincoupled receptors for high-throughput screening. Anal Biochem 314:16 –
29, 2003
25. Ding VD, Qureshi SA, Szalkowski D, Li Z, Biazzo-Ashnault DE, Xie D, Liu
K, Jones AB, Moller DE, Zhang BB: Regulation of insulin signal transduction pathway by a small-molecule insulin receptor activator. Biochem J
367:301–306, 2002
26. Cohen SM, Werrmann JG, Tota MR: 13C NMR study of the effects of leptin
treatment on kinetics of hepatic intermediary metabolism. Proc Natl Acad
Sci U S A 95:7385–7390, 1998
27. Cohen SM: 13C NMR study of effects of fasting and diabetes on the
metabolism of pyruvate in the tricarboxylic acid cycle and the utilization of
pyruvate and ethanol in lipogenesis in perfused rat liver. Biochemistry
26:581–589, 1987
28. Bogardus C, Lillioja S, Howard BV, Reaven G, Mott D: Relationships
between insulin secretion, insulin action, and fasting plasma glucose
concentration in nondiabetic and noninsulin-dependent diabetic subjects.
J Clin Invest 74:1238 –1246, 1984
29. DeFronzo RA, Ferrannini E, Simonson DC: Fasting hyperglycemia in
non-insulin-dependent diabetes mellitus: contributions of excessive hepatic glucose production and impaired tissue glucose uptake. Metabolism
38:387–395, 1989
30. Baron AD, Schaeffer L, Shragg P, Kolterman OG: Role of hyperglucagonemia in maintenance of increased rates of hepatic glucose output in type 2
diabetics. Diabetes 36:274 –283, 1987
31. MacNeil DJ, Occi JL, Hey PJ, Strader CD, Graziano MP: Cloning and
expression of a human glucagon receptor. Biochem Biophys Res Commun
198:328 –334, 1994
32. Zhang BB, Moller DE: New approaches in the treatment of type 2 diabetes.
Curr Opin Chem Biol 4:461– 467, 2000
33. Sturm NS, Lin Y, Burley SK, Krstenansky JL, Ahn JM, Azizeh BY, Trivedi D,
DIABETES, VOL. 53, DECEMBER 2004
Hruby VJ: Structure-function studies on positions 17, 18, and 21 replacement analogues of glucagon: the importance of charged residues and salt
bridges in glucagon biological activity. J Med Chem 41:2693–2700, 1998
34. Trivedi D, Lin Y, Ahn JM, Siegel M, Mollova NN, Schram KH, Hruby VJ:
Design and synthesis of conformationally constrained glucagon analogues.
J Med Chem 43:1714 –1722, 2000
35. Ahn JM, Medeiros M, Trivedi D, Hruby VJ: Development of potent glucagon
antagonists: structure-activity relationship study of glycine at position 4. J
Pept Res 58:151–158, 2001
36. Parker JC, McPherson RK, Andrews KM, Levy CB, Dubins JS, Chin JE,
Perry PV, Hulin B, Perry DA, Inagaki T, Dekker KA, Tachikawa K, Sugie Y,
Treadway JL: Effects of skyrin, a receptor-selective glucagon antagonist, in
rat and human hepatocytes. Diabetes 49:2079 –2086, 2000
37. Parker JC, Andrews KM, Allen MR, Stock JL, McNeish JD: Glycemic
control in mice with targeted disruption of the glucagon receptor gene.
Biochem Biophys Res Commun 290:839 – 843, 2002
38. Gelling RW, Du XQ, Dichmann DS, Romer J, Huang H, Cui L, Obici S, Tang
B, Holst JJ, Fledelius C, Johansen PB, Rossetti L, Jelicks LA, Serup P,
Nishimura E, Charron MJ: Lower blood glucose, hyperglucagonemia, and
pancreatic alpha cell hyperplasia in glucagon receptor knockout mice.
Proc Natl Acad Sci U S A 100:1438 –1443, 2003
39. Sloop KW, Cao JX, Siesky AM, Zhang HY, Bodenmiller DM, Cox AL, Jacobs
SJ, Moyers JS, Owens RA, Showalter AD, Brenner MB, Raap A, Gromada
J, Berridge BR, Monteith DK, Porksen N, McKay RA, Monia BP, Bhanot S,
Watts LM, Michael MD: Hepatic and glucagon-like peptide-1-mediated
reversal of diabetes by glucagon receptor antisense oligonucleotide inhibitors. J Clin Invest 113:1571–1581, 2004
40. Liang Y, Osborne MC, Monia BP, Bhanot S, Gaarde WA, Reed C, She P,
Jetton TL, Demarest KT: Reduction in glucagon receptor expression by an
antisense oligonucleotide ameliorates diabetic syndrome in db/db mice.
Diabetes 53:410 – 417, 2004
3273