1 Department of Physiology, University of British Columbia, Vancouver, British Columbia, Canada, V6T 1Z3; and 2 Department of Metabolism and Clinical Nutrition, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan
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ABSTRACT |
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The
incretins glucose-dependent insulinotropic polypeptide (GIP) and
glucagon-like peptide-1 (GLP-1) are gut hormones that act via the
enteroinsular axis to potentiate insulin secretion from the pancreas in
a glucose-dependent manner. Both GLP-1 receptor and GIP receptor
knockout mice (GLP-1R/
and
GIPR
/
, respectively) have been generated to
investigate the physiological importance of this axis. Although reduced
GIP action is a component of type 2 diabetes, GIPR-deficient mice
exhibit only moderately impaired glucose tolerance. The present study
was directed at investigating possible compensatory mechanisms that
take place within the enteroinsular axis in the absence of GIP action.
Although serum total GLP-1 levels in GIPR knockout mice were unaltered, insulin responses to GLP-1 from pancreas perfusions and static islet
incubations were significantly greater (40-60%) in
GIPR
/
than in wild-type
(GIPR+/+) mice. Furthermore, GLP-1-induced cAMP
production was also elevated twofold in the islets of the knockout
animals. Pancreatic insulin content and gene expression were reduced in
GIPR
/
mice compared with
GIPR+/+ mice. Paradoxically, immunocytochemical
studies showed a significant increase in
-cell area in the GIPR-null
mice but with less intense staining for insulin. In conclusion,
GIPR
/
mice exhibit altered islet structure
and topography and increased islet sensitivity to GLP-1 despite a
decrease in pancreatic insulin content and gene expression.
insulin; GLP-1; pancreas perfusion; cAMP; incretin
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INTRODUCTION |
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THE TERM ENTEROINSULAR AXIS was first proposed by Unger and Eisentraut (42) to describe the nutrient, neural, and endocrine interactions between the gut and the endocrine pancreas that amplify insulin secretion. The increased insulin release in response to oral glucose vs. intravenous administration is called the incretin effect. The two most important incretins that act via the enteroinsular axis are glucose-dependent insulinotropic polypeptide [GIP-(1-42)] and truncated forms of glucagon-like peptide-1 (GLP-1): GLP-1-(7-36NH2) and GLP-1-(7-37) (15, 34).
These peptides account for nearly 50% of postprandial insulin release
and therefore play a major role in glucose homeostasis (30). The insulinotropic effects of GIP and GLP-1 are
transduced through specific G protein-coupled receptors on the
-cell, resulting in stimulation of adenylyl cyclase (5,
46) and phospholipase A2 (9) and
increased levels of intracellular Ca2+ (20,
25). In addition to their insulinotropic effects, both incretins
stimulate insulin gene expression and biosynthesis in the
-cell
(5, 11). Non-
-cell-mediated effects of GLP-1, including
inhibition of gastric emptying and glucagon secretion, also contribute
to its blood glucose-lowering effects (3, 47). The
possible involvement of GIP and GLP-1 in the etiology of diabetes is
still controversial. Studies have shown that, although GIP action is
diminished, GLP-1 activity is preserved in type 2 diabetes (28,
29). A recent study from Lynn et al. (24) linked
decreased GIP action to reduced GIP receptor levels in the Vancouver
Diabetic Zucker (VDF) animal model of type 2 diabetes. Although GLP-1
secretion has been reported to be normal (13, 29, 44),
increased (16, 32), or decreased (43) in type
2 diabetes due to its preserved insulinotropic activity in the diabetic
state, GLP-1 has been widely studied as a possible therapeutic agent
(17).
The relative contribution of the incretins to the enteroinsular axis is
still under investigation. Mice that are deficient in functional GIP or
GLP-1 receptors (GIPR/
and
GLP-1R
/
mice, respectively) provide unique
models for the study of incretin physiology. Both mouse models have
impaired glucose tolerance but normal feeding behavior and body weight
(26, 37). Different fasting plasma glucose levels have
been reported in different colonies. We report that the
GLP-1R
/
colony exhibits normal glucose
levels, whereas others have reported increased fasting levels
(33, 37). In addition, GLP-1R
/
mice have elevated plasma GIP levels and increased
-cell sensitivity to GIP, demonstrating that disruption of one component of the enteroinsular axis may be compensated for by another (33).
We hypothesized that this compensatory mechanism is due to a
physiological balance between the incretins, allowing for maintenance
of glucose homeostasis, and that a similar compensation (enhanced
GLP-1/insulin axis) would be observed in the
GIPR
/
mouse. In the present study, we have
tested this hypothesis and report that the sensitivity of the
-cell
to GLP-1 is indeed enhanced in the GIPR
/
mouse along with reduced pancreatic insulin content and altered islet topography.
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MATERIALS AND METHODS |
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Animals.
The background and generation of GIPR-deficient C57BL/6 mice used in
this study have been previously described (26). All animal
experiments were performed in accordance with the guidelines put forth
by the University of British Columbia Committee on Animal Care and the
Canadian Council on Animal Care. For all the experiments, only 9- to
14-wk-old male GIPR/
and C57BL/6 control
mice were used.
Oral glucose tolerance test and hormone radioimmunoassays.
After an overnight fast, control and GIPR/
mice were administered 1 g/kg glucose (as a 10% solution) via gavage.
Blood glucose levels were measured individually for each mouse by use
of a handheld glucometer (Surestep; Lifescan, Burnaby, BC, Canada).
Because only a limited volume of blood could be removed from each mouse and it was necessary to measure plasma GIP, GLP-1, and insulin levels
in each sample, blood from 5-6 animals was pooled at
t = 0 and t = 20 min after an oral
glucose tolerance test (OGTT). Plasma was then separated by
centrifugation at 12,000 g for 15 min at 4°C and stored at
20°C until hormone radioimmunoassay (RIA). The GIP RIA utilized a
COOH-terminally directed antibody (rabbit anti-human) that has been
extensively used for measuring total GIP immunoreactivity
[GIP-(1-42) and GIP-(3-42)]
(33). Total GLP-1 concentrations were measured by using
the rat total GLP-1 kit from Linco (St. Charles, MO) as directed by the
manufacturer. The GLP-1 antibody recognizes total immunoreactive GLP-1
(GLP-1-(7-36NH2), GLP-1-(7-37), and
NH2-terminally truncated forms) and has a detection limit
of 3 pM. Insulin was assayed by using a sensitive rat insulin kit
(Linco) with an antibody that cross-reacts 100% with mouse insulin.
Plasma dipeptidyl peptidase IV (DP IV) activity (µU/ml) was measured
colorimetrically (405 nm) through enzymatic release of
p-nitroanilide (p-NA) from
H-gly-pro-p-NA (35).
Insulin tolerance test.
GIPR+/+ and GIPR/
mice were fasted for 3 h and given 0.01 U/g insulin (Humulin R,
100 U/ml; Lilly, Toronto, ON, Canada) by intraperitoneal injection.
Blood glucose was measured with a handheld glucometer.
In vitro perfused pancreas.
GIPR+/+ and GIPR/
mice were fasted overnight and anesthetized by intraperitoneal
injection of 80 mg/kg pentobarbital sodium (Somnotol; MTC
Pharmaceuticals, Cambridge, ON, Canada) before surgery. The surgical
procedure and the pancreas perfusion protocol are described in Pederson
et al. (33). Briefly, the abdominal aorta and the portal
vein were cannulated with PE-50 tubing (Cole-Palmer, Chicago, IL). The
perfusate consisted of a Krebs-Ringer bicarbonate buffer supplemented
with 3% dextran (Sigma, Oakville, ON, Canada) and 0.2% bovine
serum albumin (BSA, fraction V, RIA grade; Sigma) gassed with
5% CO2 to achieve and maintain pH 7.4. The flow rate was
maintained at 1 ml/min, and the outflow was collected at 1-min intervals. GIP and GLP-1 (both from Probiodrug, Hala, Germany) were
delivered by sidearm infusion in the presence of 16.7 mM glucose,
resulting in a 1 nM final concentration, whereas arginine (10 mM;
Sigma) was mixed directly into the perfusate containing 8.8 mM glucose.
Insulin was measured by RIA as previously described (33).
Isolation and culture of pancreatic islets. Fed mice were anesthetized with Somnotol, and islets of Langerhans were isolated by collagenase digestion. Collagenase (type V, 2 mg/ml; Sigma) in Hanks' balanced salt solution (HBSS; GIBCO-Life Technologies, Burlington, ON, Canada) supplemented with 10 mM HEPES (GIBCO), 2 mM L-glutamine (Sigma), and 0.2% BSA was injected into the common bile duct to inflate the pancreas. The pancreas was then removed and digested at 37°C for 10 min. The pancreas was then dispersed mechanically with a siliconized Pasteur pipette, washed, filtered through a 1-mm nylon screen, and washed again. Islets were separated by centrifugation at 1,800 g for 20 min in a discontinuous dextran gradient. Hand-picked islets were cultured overnight in RPMI 1640 (Sigma) with 8.8 mM glucose, 10% fetal calf serum (Cansera, Rexdale, ON, Canada), 50 U/ml each of penicillin G (Sigma) and streptomycin (Sigma), 0.07% human serum albumin (Sigma), 0.0025% human apotransferrin (GIBCO), 25 pM sodium selenite (VWR, Mississauga, ON, Canada) and 10 µM ethanolamine (VWR).
Islet cAMP and insulin measurements.
After overnight culture, 15-18 healthy islets were selected,
washed twice with 0.5 ml of Krebs-Ringer supplemented with 0.2% BSA
and 4.4 mM glucose, and incubated at 37°C for 30 min. Thereafter, islets were incubated in the presence or absence of 10 nM GIP or GLP-1
or 10 µM forskolin (Sigma) in the same buffer with 0.5 mM
3-isobutyl-1-methylxanthine (Research Biochemicals International, Natick, MA) and 16.7 mM glucose. After a 30-min incubation, islets were
lysed by boiling for 3 min in 0.05 N HCl. Samples were subsequently dried by vacuum centrifugation (Speed-Vac; Sorvall, Farmingdale, NY)
and stored at 20°C. cAMP levels were assayed by using a kit according to the manufacturer's instructions (Biomedical Technologies, Stoughton, MA). For insulin measurements, islets were collected after
overnight culture, washed twice with Krebs-Ringer, and incubated for 45 min in 4.4 mM glucose containing Krebs-Ringer supplemented with 0.1%
BSA. After a short centrifugation, the medium was replaced with either
16.7 mM glucose alone or 16.7 mM glucose plus 10 nM GIP or GLP-1. After
45 min, islets were lysed by boiling for 5 min in 1 M acetic acid and
centrifuged, and supernatants were assayed for insulin
(24).
Measurement of pancreatic insulin content.
Animals were rendered unconscious with CO2 and
exsanguinated. Pancreata were removed, homogenized in 2 M acetic acid,
and then boiled for 5 min. Homogenates were centrifuged at 15,000 g for 15 min, and the supernatant was stored at 70°C.
Total protein levels were measured with a bicinchoninic acid kit
(Pierce, Rockford, IL), and insulin values (measured as described) were
normalized to total protein content (ng/µg protein)
(33).
Isolation and measurement of islet insulin and GLP-1 receptor
mRNA by reverse transcriptase real-time polymerase chain reaction
(PCR).
Freshly isolated islets were washed twice with HBSS and solubilized in
1 ml TRIzol (GIBCO) and kept at 70°C. After isolation, 50 ng of
total RNA were subjected to reverse transcription (RT) in a volume of
10 µl containing 0.5 mM deoxynucleotide triphosphates, 15 pmol
specific primers targeted at the carboxy termini of the mouse GLP-1
receptor open reading frame (5'-ACC AAC AGG GAG GAC CGG-3') or the
mouse insulin II gene (5'-GTA GTT CTC CAG CTG GTA GAG GG-3') 100 U
Superscript II RNAse H
reverse transcriptase (GIBCO), 10 U RNAse inhibitor (RNA Guard; Amersham Pharmacia, Piscataway, NJ), 1 mM
dithiothreitol, 50 mM Tris · HCl (pH 8.3), 75 mM
KCl, and 3 mM MgCl2. After RT, 10 ng (2 µl) of mouse cDNA
were used in the real-time PCR reaction to measure insulin and GLP-1
receptor expression. The PCR reaction mix consisted of 1× TaqMan
Buffer A (PE Applied Biosystems, Foster City, CA), 10 mM
MgCl2, 200 µM each dATP, dCTP, and dGTP, 400 µM dUTP,
200 nM mouse GLP-1 receptor 5' forward primer (5'-CAG GGC TTG ATG GTG
GCT ATC-3') or mouse insulin II 5' forward primer (5'-TGG AGG CCC GGG
AGC-3'), 200 nM mouse GLP-1 receptor 3' reverse primer (5'-CGC TCC CAG
CAT TTC CG-3') or mouse insulin II 3' reverse primer (5'-ATC TAC AAT
GCC ACG CTT CTG C-3'), and 100 nM GLP-1 receptor probe colabeled with
the fluorescent dyes VIC and 6-carboxy-tetramethylrhodamine (TAMRA)
(5'-ACT GCT TTG TCA ACA ATG AGG TCC AGA TGG-3') or insulin II probe
colabeled with fluorescent dyes TET and TAMRA (5'-ACC TTC AGA CCT TGG
CAC TGG AGG TG-3'), 0.01 U/µl AmpErase-uracil N-glycosylase (UNG; PE Applied Biosystems), and 0.025 U/µl
AmpliTaq Gold (PE Applied Biosystems). PCR reactions were
carried out in triplicate in the PE Applied Biosystems 7700 sequence
detection system. The reaction profile included a 10-min preincubation
at 50°C to allow the UNG to degrade any uracil-containing nucleic acids and a further 10-min incubation at 94°C to activate the AmpliTaq Gold. After these preincubations, a two-step PCR protocol was
carried out, which included a denaturation step at 94°C for 15 s
followed by a 1-min annealing/extension step at 60°C. Fluorescence was measured during the annealing/extension steps over 40 cycles and
used to calculate a cycle threshold (Ct), i.e., the point at which the
reaction is in the exponential phase and is detectable by the hardware.
All reactions followed the typical sigmoidal reaction profile, and Ct
was used as a measure of amplicon abundance (12). The
results were normalized over total wild-type mRNA levels.
Immunocytochemistry.
Mice were fasted overnight before being killed. Pancreata from
wild-type and knockout mice (n = 5 each) were
fixed separately in Bouin's solution (75% picric acid, 8%
formaldehyde, 5% glacial acetic acid) for 1 h at room temperature
and washed thoroughly with 70% ethanol. The tissue was embedded in
paraffin wax, and three consecutive 5-µm sections were taken 300 µm
apart and mounted on glass slides. The study was carried out blind:
slides were coded to prevent identification of +/+ and
/
tissues before quantification. The sections were
dewaxed in xylene and cleared in petroleum ether (Sigma). The sections
were then rehydrated in PBS (80 mM Na2HPO4,
1.47 mM KH2PO4, 2.86 mM KCl, 137 mM NaCl). To
control for intraimmunostain variability, all of the sections were
incubated in the same batch of solutions and stained simultaneously. The
-cells were detected by overnight incubation with a polyclonal rabbit anti-mouse insulin antibody (1:100, Santa Cruz Biotechnologies, Santa Cruz, CA), followed by a 90-min incubation with
a biotinylated goat anti-rabbit secondary antibody (1:300, Jackson
Labs, West Grove, PA) at room temperature. Sections were then washed
and incubated with avidin-biotin peroxidase complex (Vector Labs, Burlington, ON, Canada) at a dilution of 1:1,000 in PBS
supplemented with 5% horse serum (Sigma). The peroxidase reaction was
developed with 2% diaminobenzidine tetrahydrochloride in 0.05 M Tris
(pH 7.5) with 0.2% H2O2. After being
counterstained with hematoxylin for 5 min, the sections were dehydrated
through graded alcohol, and coverslips were applied with Permount
(Fisher Scientific, Nepean, ON, Canada). The sections were analyzed
using the NIH Image software (http://rsb.info.nih.gov/nih-image/), and
data were analyzed as islet area over total pancreatic area. Five
separate fields of view (under ×10 magnification) per section were
randomly chosen, the periphery of the islets in this area was outlined, and area was determined by using the analysis software. The islet area
from the five random samples was then normalized to the total pancreatic area in those five fields. Once the quantification had been
completed, the source of the sections (GIPR+/+
or GIPR
/
) was identified and statistical
significance was assessed.
Data analysis.
All data are expressed as means ± SE. An unpaired Student's
t-test and a Mann-Whitney U-test (exclusively for
immunocytochemistry) were used to compare the control values with
GIPR/
mouse values, where P < 0.05 was considered statistically significant. The data analysis and
area under the curve calculations were carried out by using graphic
analysis software (Graphpad, Prism, San Diego, CA).
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RESULTS |
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OGTT.
The OGTT profile of GIPR/
mice over 120 min
is very similar to that of the wild-type control animals (Fig.
1A). The 15- and 30-min blood
glucose were moderately (18%) but significantly greater in
GIPR
/
mice. To allow for concurrent
measurement of insulin and incretin levels, basal and 20-min blood
samples from another OGTT were pooled. This time point was chosen on
the basis of preliminary studies to achieve near-peak incretin levels
(data not shown). Fasting glucose and insulin levels were both
comparable in GIPR
/
and wild-type mice (Fig.
1, B and C). However, after a glucose challenge,
the 20-min plasma glucose levels were significantly higher in
GIPR
/
mice than in wild-type mice
(P < 0.05), and insulin levels were 45% lower in
GIPR
/
mice than in wild-type mice (Fig. 1,
B and C). To further assess the hormonal
components of the enteroinsular axis in
GIPR
/
mice, fasting and 20-min total plasma
immunoreactive GLP-1 and immunoreactive GIP levels were determined
(Fig. 1, D and E). Total GLP-1 levels did not
differ in GIPR
/
mice compared with wild-type
(Fig. 1D, P > 0.05). However, fasting plasma GIP levels were elevated, whereas the 20-min levels were lowered
by 25% in GIPR
/
compared with
GIPR+/+ mice (Fig. 1E,
P < 0.05). Plasma DP IV activity was
unaltered between groups: values for fasting were 10.5 ± 0.9 vs.
10.2 ± 0.8 µU/ml and 9.1 ± 1.5 vs. 8.7 ± 0.6 µU/ml for 20-min in GIPR+/+ and
GIPR
/
mice, respectively.
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Insulin tolerance test.
To investigate the insulin sensitivity of the
GIPR/
mice, blood glucose levels were
determined after an insulin challenge. Blood glucose profile over
1 h did not differ in GIPR
/
mice
compared with wild-type control mice (Fig.
2).
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In vitro insulin responses to GIP, glucose, arginine, and GLP-1.
To verify that GIP was unable to stimulate insulin secretion from the
pancreata of GIPR/
animals, in vitro
perfusions were carried out (Fig.
3A). As expected, 1 nM GIP
generated no insulin response from the perfused knockout pancreata.
Subsequently, low (4.4 mM) and high (16.7 mM) glucose perfusions were
also performed, demonstrating that glucose-stimulated insulin
secretions in GIPR
/
and wild-type mice were
comparable (Fig. 3B). In type 2 diabetic patients, insulin
responses to secretagogues such as high glucose, sulfonylurea, and
arginine are blunted (45). To assess the
insulin response of GIPR
/
islets to a
stimulant other than glucose, high-dose arginine (10 mM) perfusions
were performed in the presence of 8.8 mM glucose (Fig. 3C).
The results showed no significant differences in insulin secretion
between GIPR
/
and control animals. However,
GIPR
/
mice exhibited a higher peak and
sustained insulin release in response to GLP-1 perfusion (Fig.
3D). The integrated insulin response to perfusion with 1 nM
GLP-1 was 60% greater in GIPR
/
mice
compared with wild-type mice (Fig. 3E). To determine whether these results were due to an inherent change in islet physiology, islets were isolated, cultured overnight, and stimulated with low (4.4 mM) and high (16.7 mM) glucose alone or in the presence of either 10 nM
GIP or GLP-1 (Fig. 4A). The
insulin secreted over 45 min was 40% greater in response to GLP-1
stimulation for GIPR
/
vs.
GIPR+/+ islets (P < 0.05),
consistent with the data from perfusion experiments. Additionally, GIP did not stimulate insulin release from
GIPR
/
islets, and the insulin response to
16.7 mM glucose was comparable in both groups.
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Intracellular cAMP production in isolated islets.
To correlate GLP-1-stimulated insulin release with receptor activation,
we measured cAMP production in response to GLP-1 in isolated islets
(Fig. 4B). Forskolin was used as a positive control to
assess maximal cAMP production and thus allow normalization of
responses to account for discrepancies in islet size and number. Interestingly, basal (16.7 mM glucose) cAMP levels were significantly increased in GIPR/
mice compared with
wild-type mice. GLP-1-stimulated cAMP production was
also significantly increased in GIPR
/
animals vs. wild-type mice (P < 0.05), implying
that
-cell sensitivity to GLP-1 was increased. Thus these findings
are also consistent with the perfusion and static islet stimulation experiments.
Pancreatic insulin content and islet insulin and GLP-1 mRNA
content.
Both GIP and GLP-1 stimulate insulin gene transcription and protein
synthesis in the -cell (11). Thus absence of GIP action may lead to alterations in insulin gene transcription and, therefore, pancreatic insulin content. The total insulin content from fed mice
pancreata was significantly lower (~40%) in
GIPR
/
than in GIPR+/+
mice (Fig. 6A; P < 0.05). Furthermore,
these data are supported by the finding that insulin mRNA levels were
significantly reduced (~40%) in isolated islets of
GIPR
/
mice compared with controls (Fig.
5A, P < 0.05). Finally, assessment of GLP-1
receptor mRNA levels revealed that, despite an increase in GLP-1
sensitivity, there was no increase in GLP-1 receptor mRNA levels in the
islets of GIPR
/
mice (Fig. 5B).
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Immunocytochemistry.
Immunocytochemical studies were carried out to assess the effect of GIP
receptor deficiency on islet and pancreas morphology. -Cell area as
a percentage of total pancreatic area was significantly increased
(~45%) in knockout vs. wild-type mice (Fig. 6B;
P < 0.05). Additionally,
when stained under identical experimental conditions, the staining
intensity for insulin was reduced in GIPR
/
islets (Fig. 6, C and D). The whole pancreas
weight was not different between groups: 1.6 ± 0.4 vs. 1.7 ± 0.3 g in GIPR+/+ and
GIPR
/
, respectively.
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DISCUSSION |
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The reduced effect of GIP on insulin secretion in type 2 diabetic patients has been described (21, 29). It has been
suggested that this might be due to reduced receptor expression in the
-cell, resulting in its lowered sensitivity to GIP
(18). Recently, this possibility was addressed in a model
of type 2 diabetes, the VDF rat, in which it was demonstrated that GIP
receptor expression was reduced (24). Therefore, mice with
a targeted disruption of the GIPR gene
(GIPR
/
) may provide a useful model for
studying the potential implications of a lack of GIP signaling on
glucose homeostasis and the development of type 2 diabetes. Miyawaki et
al. (26) have shown that these mice exhibit modest glucose
intolerance along with a 50% reduction in insulin secretion in
response to oral glucose, whereas weight gain was reported to remain
unchanged under both normal and high-fat diet conditions. It was the
hypothesis of the present study that these mice would exhibit
compensatory changes in the enteroinsular axis to overcome the absence
of GIP action.
Immunoneutralization studies examining the relative contribution of GIP
to the enteroinsular axis were first reported in the early 1980s
(6-8). In more recent studies, the treatment of rats with a GIP antagonist, GIP-(7-30NH2), has been shown
to result in a 72% decrease in postprandial insulin release along with
normal glucose levels (40). Later, the same group provided
evidence that GIP-(7-30NH2) inhibits glucose transport
from the small intestine, which might in part explain the relatively
small rise in serum glucose levels after oral glucose despite a
profound decrease in postprandial insulin levels (41).
Studies using a GIPR antibody have suggested that GIP acts as an
anticipatory signal to the -cell to potentiate insulin release,
which in turn primes the periphery for glucose disposal
(22). The same antibody was used on
GLP-1R
/
and GLP-1R+/+
mice, and it was concluded by Baggio et al. (2) that GIP
had a restricted role in the regulation of glucose homeostasis. These studies involved relatively short-term antagonist administration and
therefore may not be reflective of the consequences of the chronic
absence of the GIP action. The use of GIPR-null mice provides a new
approach to the investigation of the effects of GIP.
Despite the loss of GIP receptors, the 2-h blood glucose profiles for
GIPR/
and wild-type mice are remarkably
similar (Fig. 1A). Blood glucose reached peak values for
both groups of animals at the 15-min time point, and the curves merged
after 40 min. The subtle increase (18%) in plasma glucose levels at
the 20th min of the OGTT does not correlate with the more profound
reduction (55%) in plasma insulin levels in the
GIPR
/
animals (Fig. 1, B and
C), suggesting a possible change in insulin sensitivity or
glucose disposal. Controversy exists as to whether GLP-1 is capable of
exerting insulin-like effects on peripheral tissue in addition to the
well-studied insulinotropic effects. Studies have shown that GLP-1
improves glucose disposal in type 2 diabetes by enhancing
insulin-stimulated glucose utilization (36), and these
effects have been shown to be independent of the amount of insulin
secreted (4, 14). Recently, Ahren and Pacini
(1) reported that, in mice, the effects of GLP-1 on glucose homeostasis were mainly insulin mediated and the use of a
relatively specific GLP-1 receptor antagonist,
exendin-(9-39), reversed these actions. It
remains unclear whether an increased extrapancreatic sensitivity to
GLP-1 might play a role in the alteration in glucose clearance in
GIPR
/
mice. We report normal sensitivity to
insulin in GIPR
/
mice (Fig. 2); therefore, it appears
that enhanced GLP-1 action is not insulin mediated. However, GLP-1 is
able to potentiate insulin action (36), and it is possible
that, in GIPR
/
mice, enhanced GLP-1 action
ameliorates insulin action. Furthermore, it is also possible that, in
these mice, the enhanced glucose disposal (relative to the insulin
levels present) could be a result of the delayed gastric emptying
(caused by GLP-1) (31) due to increased peripheral tissue
GLP-1 sensitivity.
Similar to GIPR/
mice,
GLP-1R
/
mice have been shown to exhibit
modest glucose intolerance, with upregulation, in this case, of the GIP
component of the enteroinsular axis (33). The present study was designed in part to test whether the converse was true in GIPR
/
animals. Miyawaki et al.
(26) showed that, when administered an
intraperitoneal glucose challenge (thus bypassing the enteroinsular axis), GIPR
/
mice exhibited no
alteration in glucose disposal relative to wild-type animals. In the
present study, pancreas perfusion (Fig. 3), static islet stimulation
(Fig. 4A), and cAMP production (Fig. 4B) data
clearly showed that the GLP-1 component of the enteroinsular axis in
GIPR
/
mice was upregulated. These data agree
with the evidenced alteration in oral glucose tolerance in the face of
unchanged intraperitoneal glucose tolerance. The combined findings that
in vitro insulin responses to high glucose and arginine are similar and
that islet GLP-1 receptor mRNA levels in
GIPR
/
and in GIPR+/+
mice are comparable suggest that compensation occurs distally to the
GLP-1 receptor on the
-cell. Because both incretins act through G
protein-coupled receptors and signal via the adenylyl cyclase-cAMP
system (5, 27, 46), it could be hypothesized that the
permanent absence of GIP receptors leads to a compensatory increase in
coupling efficiency of GLP-1 receptors. Although the GLP-1 receptor
mRNA levels are comparable, we do not have information about the
protein synthesis. Hence, despite similar gene expression, the protein
synthesis might be enhanced, leading to increased sensitivity to GLP-1.
The possibility of upregulation of GLP-1 secretion appears unlikely,
since no changes in GLP-1 levels were observed 20 min after an oral
glucose challenge (Fig. 1D), nor were there changes in
activity of plasma DP IV, the primary inactivating enzyme for GIP and
GLP-1 (35). Together these data suggest that the majority
of the compensatory changes in the GLP-1 axis of GIPR
/
mice lay at a postreceptor level in
the
-cell. That said, the observed reduction in circulating insulin
levels and modest increase in blood glucose show that compensation by
the GLP-1 axis is not complete and that, although the functions of GIP
and GLP-1 overlap, both are required for proper glycemic control.
GIPR/
mice were shown to have a 40%
reduction in pancreatic insulin content and gene expression concomitant
with a twofold increase in
-cell area (Fig. 6, A and
B), suggesting that insulin gene expression and content were
reduced in the GIPR-null mice on a cellular level. Because both GIP and
GLP-1 stimulate insulin gene expression (5, 11), it might
be predicted that an increase in
-cell GLP-1 sensitivity in the
absence of GIP action could protect against a decrease in islet insulin
mRNA and protein levels. The decrease in insulin mRNA and protein
synthesis in GIPR
/
mice is comparable to the
35% decrease that was shown by Pederson et al. (33) in
GLP-1R
/
mice. Hence, absence of either of
the incretins results in abnormalities within the
-cell, leading to
impaired insulin content. Thus the compensation by GLP-1 or by GIP at
the
-cell level, in GIPR
/
and in
GLP-1R
/
mice, respectively, seems not to
extend as far as insulin biosynthesis.
In addition to stimulation of insulin production on the cellular level,
GLP-1 has also been shown to be involved in the morphological development of the islets of Langherhans. In immunocytochemical studies, Ling et al. (23) showed - and
-cell
migration toward the islet core and a reduction in islet size in
GLP-1R
/
mouse pancreata. Examination of
pancreatic sections from GIPR
/
mice with the
same objective showed no such changes in endocrine cell distribution,
only in
-cell area (Fig. 6). Reduced insulin gene expression and
insulin content correlated well with less intensely stained islets in
GIPR
/
mice (Fig. 6). Immunostaining for
glucagon and somatostatin showed normal topology and distribution with
no indication of migration toward the islet core (data not shown). Very
recently, it has been shown that GLP-1 has growth hormone-like effects
on pancreatic islets and on
-cells (19). Thus the
enhanced GLP-1 action on the GIPR
/
mouse
pancreas, indicated by pancreas perfusions and static islet stimulation, was consistent with the observed increase in
-cell area. Recently, studies have shown on
TC3 insulin-secreting tumors that GIP is a regulator of upstream kinases of apoptosis
cascades. In this regard, our finding of increased
-cell area in
GIPR
/
mice is not fully understood
(10, 38, 39). Further studies are required to examine the
role of GIP on islet growth and insulin gene expression to clarify
these findings. To date, there have been no conclusive studies that
have examined the effects of GIP on islet/
-cell development and
survival in vivo.
In summary, we have demonstrated that disruption of the GIP component
of the enteroinsular axis in mice results in decreased insulin gene
transcription and protein biosynthesis, increased islet sensitivity to
GLP-1, and changes in islet structure. We report that compensation for
the absence of a functional GIP receptor occurs, in part, by
upregulation of the GLP-1 component of the enteroinsular axis. The
physiological changes that take place in both the
GIPR/
and the
GLP-1R
/
strains of knockout mice suggest
that the incretins act in concert to maintain glucose homeostasis and
that a balance between the two is required for proper function of the
enteroinsular axis. Further experiments, targeted at clarifying the
molecular changes that occur within the
-cell, are required to
extend our understanding of GIP physiology.
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ACKNOWLEDGEMENTS |
---|
Technical assistance was provided by Cuilan Nian.
![]() |
FOOTNOTES |
---|
This work was supported by the Canadian Institutes for Health Research Grant 590007, and the Canada Foundation for Innovation.
Address for reprint requests and other correspondence: Raymond A. Pederson, Dept. of Physiology, Faculty of Medicine, Univ. of British Columbia, 2146 Health Sciences Mall, Vancouver, BC, Canada V6T 1Z3 (E-mail: pederson{at}interchange.ubc.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published January 21, 2003;10.1152/ajpendo.00270.2002
Received 19 June 2002; accepted in final form 18 January 2003.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Ahren, B,
and
Pacini G.
Dose-related effects of GLP-1 on insulin secretion, insulin sensitivity, and glucose effectiveness in mice.
Am J Physiol Endocrinol Metab
277:
E996-E1004,
1999
2.
Baggio, L,
Kieffer TJ,
and
Drucker DJ.
Glucagon-like peptide-1, but not glucose-dependent insulinotropic peptide, regulates fasting glycemia and nonenteral glucose clearance in mice.
Endocrinology
141:
3703-3709,
2000
3.
Creutzfeldt, WOC,
Orskov C,
Kleine N,
Holst JJ,
Willms B,
and
Nauck MA.
Glucagonostatic actions and reduction of fasting hyperglycemia by exogenous glucagon-like peptide-1 (7-36) amide in type 1 diabetic patients.
Diabetes Care
19:
580-586,
1996[Abstract].
4.
D'Alessio, DA,
Prigeon RL,
and
Ensinck JW.
Enteral enhancement of glucose disposition by both insulin- dependent and isulin-independent processa physiological role of glucagon-like peptide 1.
Diabetes
44:
1433-1437,
1995[Abstract].
5.
Drucker, DJ,
Philippe J,
Mojsov S,
Chick WL,
and
Habener JF.
Glucagon-like peptide I stimulates insulin gene expression and increases cAMP levels in a rat islet cell line.
Proc Natl Acad Sci USA
84:
3434-3438,
1987[Abstract].
6.
Ebert, R,
and
Creutzfeldt W.
Influence of gastric inhibitory polypeptide antiserum on glucose-induced insulin secretion in rats.
Endocrinology
111:
1601-1606,
1982[Abstract].
7.
Ebert, R,
Illmer K,
and
Creutzfeldt W.
Release of gastric inhibitory polypeptide (GIP) by intraduodenal acidification in rats and humans and abolishment of the incretin effect of acid by GIP-antiserum in rats.
Gastroenterology
76:
515-523,
1979[ISI][Medline].
8.
Ebert, R,
Unger H,
and
Creutzfeldt W.
Preservation of incretin activity after removal of gastric inhibitory polypeptide (GIP) from rat gut extracts by immunoadsorption.
Diabetologia
24:
449-454,
1983[ISI][Medline].
9.
Ehses, JA,
Lee SST,
Pederson RA,
and
McIntosh CHS
A new pathway for glucose-dependent insulinotropic polypeptide (GIP) receptor signalingevidence for the involvement of phospholipase A, in GIP-stimulated insulin secretion.
J Biol Chem
276:
23667-23673,
2001
10.
Ehses, JA,
Pelech SL,
Pederson RA,
and
McIntosh CH.
Glucose-dependent insulinotropic polypeptide activates the Raf-Mek1/2-ERK1/2 module via a cyclic AMP/cAMP-dependent protein kinase/Rap1-mediated pathway.
J Biol Chem
277:
37088-37097,
2002
11.
Fehmann, HC,
Gherzi R,
and
Goke B.
Regulation of islet hormone gene expression by incretin hormones.
Exp Clin Endocrinol Diabetes
103:
56-65,
1995[ISI][Medline].
12.
Freeman, WM,
Walker SJ,
and
Vrana KE.
Quantitative RT-PCR: pitfalls and potential.
Biotechniques
26:
112-125,
1999[ISI][Medline].
13.
Greenbaum, C,
Prigeon R,
and
D'Alessio D.
Impaired -cell function, incretin effect, and glucagon suppression in patients with type 1 diabetes who have normal fasting glucose.
Diabetes
51:
951-957,
2002
14.
Gutniak, MK,
Svartberg J,
Hellstrom PM,
Holst JJ,
Adner N,
and
Ahren B.
Antidiabetogenic action of glucagon-like peptide-1 related to administration relative to meal intake in subjects with type 2 diabetes.
J Intern Med
250:
81-87,
2001[ISI][Medline].
15.
Habener, JF.
The incretin notion and its relevance to diabetes.
Endocrinol Metab Clin North Am
22:
775-794,
1993[ISI][Medline].
16.
Hiroyoshi, M,
Tateishi K,
Yasunami Y,
Maeshiro K,
Ono J,
Matsuoka Y,
and
Ikeda S.
Elevated plasma levels of glucagon-like peptide-1 after oral glucose ingestion in patients with pancreatic diabetes.
Am J Gastroenterol
94:
976-981,
1999[ISI][Medline].
17.
Holst, JJ.
Glucagon-like peptide-1, a gastrointestinal hormone with a pharmaceutical potential.
Curr Med Chem
6:
1005-1017,
1999[ISI][Medline].
18.
Holst, JJ,
Gromada J,
and
Nauck MA.
The pathogenesis of NIDDM involves a defective expression of the GIP receptor.
Diabetologia
40:
984-986,
1997[Medline].
19.
Holz, GG,
and
Leech CA.
Glucagon-like peptide-1: an insulinotropic hormone with potent growth factor actions at the pancreatic islets of Langerhans.
In: Molecular Basis of Pancreas Development and Function, edited by Habener JF,
and Hussain MA.. Norwell, MA: Kluwer Academic, 2001, p. 109-141.
20.
Kieffer, TJ,
and
Habener JF.
The glucagon-like peptides.
Endocr Rev
20:
876-913,
1999
21.
Krarup, T,
Saurbrey N,
Moody AJ,
Kuhl C,
and
Madsbad S.
Effect of porcine gastric inhibitory polypeptide on -cell function in type I and type II diabetes mellitus.
Metabolism
36:
677-682,
1984.
22.
Lewis, JT,
Dayanandan B,
Habener JF,
and
Kieffer TJ.
Glucose-dependent insulinotropic polypeptide confers early phase insulin release to oral glucose in rats: demonstration by a receptor antagonist.
Endocrinology
141:
3710-3716,
2000
23.
Ling, Z,
Wu D,
Zambre Y,
Flamez D,
Drucker DJ,
Pipeleers DG,
and
Schuit FC.
Glucagon-like peptide 1 receptor signaling influences topography of islet cells in mice.
Virchows Arch
438:
382-387,
2001[ISI][Medline].
24.
Lynn, FC,
Pamir N,
Ng EHC,
McIntosh CHS,
Kieffer TJ,
and
Pederson RA.
Defective glucose-dependent insulinotropic polypeptide receptor expression in diabetic fatty Zucker rats.
Diabetes
50:
1004-1011,
2001
25.
McIntosh, CH,
Wheeler MB,
Gelling RW,
Brown JC,
and
Pederson RA.
GIP receptors and signal-transduction mechanisms.
Acta Physiol Scand
157:
361-365,
1996[ISI][Medline].
26.
Miyawaki, K,
Yamada Y,
Yano H,
Niwa H,
Ban N,
Ihara Y,
Kubota A,
Fujimoto S,
Kajikawa M,
Kuroe A,
Tsuda K,
Hashimoto H,
Yamashita T,
Jomori T,
Tashiro F,
Miyazaki J,
and
Seino Y.
Glucose intolerance caused by a defect in the entero-insular axis: a study in gastric inhibitory polypeptide receptor knockout mice.
Proc Natl Acad Sci USA
96:
14843-14847,
1999
27.
Moens, K,
Heimberg H,
Flamez D,
Huypens P,
Quartier E,
Ling Z,
Pipeleers D,
Gremlich S,
Thorens B,
and
Schuit F.
Expression and functional activity of glucagon, glucagon-like peptide I, and glucose-dependent insulinotropic peptide receptors in rat pancreatic islet cells.
Diabetes
45:
257-261,
1996[Abstract].
28.
Nauck, M,
Stockmann F,
Ebert R,
and
Creutzfeldt W.
Reduced incretin effect in type 2 (non-insulin-dependent) diabetes.
Diabetologia
29:
46-52,
1986[ISI][Medline].
29.
Nauck, MA,
Heimesaat MM,
Orskov C,
Holst JJ,
Ebert R,
and
Creutzfeldt W.
Preserved incretin activity of glucagon-like peptide 1 [7-36 amide] but not of synthetic human gastric inhibitory polypeptide in patients with type-2 diabetes mellitus.
J Clin Invest
91:
301-307,
1993[ISI][Medline].
30.
Nauck, MA,
Homberger E,
Siegel EG,
Allen RC,
Eaton RP,
Ebert R,
and
Creutzfeldt W.
Incretin effects of increasing glucose loads in man calculated from venous insulin and C-peptide responses.
J Clin Endocrinol Metab
63:
492-498,
1986[Abstract].
31.
Nauck, MA,
Niedereichholz U,
Ettler R,
Holst JJ,
Orskov C,
Ritzel R,
and
Schmiegel WH.
Glucagon-like peptide 1 inhibition of gastric emptying outweighs its insulinotropic effects in healthy humans.
Am J Physiol Endocrinol Metab
273:
E981-E988,
1997
32.
Orskov, C,
Jeppesen J,
Madsbad S,
and
Holst JJ.
Proglucagon products in plasma of noninsulin-dependent diabetics and nondiabetic controls in the fasting state and after oral glucose and intravenous arginine.
J Clin Invest
87:
415-423,
1991[ISI][Medline].
33.
Pederson, RA,
Satkunarajah M,
McIntosh CHS,
Scrocchi LA,
Flamez D,
Schuit F,
Drucker DJ,
and
Wheeler MB.
Enhanced glucose-dependent insulinotropic polypeptide secretion and insulinotropic action in glucagon-like peptide 1 receptor (/
) mice.
Diabetes
47:
1046-1052,
1998[Abstract].
34.
Pederson, RA,
Schubert HE,
and
Brown JC.
The insulinotropic action of gastric inhibitory polypeptide.
Can J Physiol Pharmacol
53:
217-223,
1975[ISI][Medline].
35.
Pospisilik, JA,
Stafford SG,
Demuth H-U,
Brownsey R,
Parkhouse W,
Finegood DT,
McIntosh CHS,
and
Pederson RA.
Long-term treatment with dipeptidyl peptidase IV inhibitor P32/98 causes sustained improvements in glucose tolerance, insulin sensitivity, hyperinsulinemia, and beta cell glucose responsiveness in VDF (fa/fa) Zucker rats.
Diabetes
51:
943-950,
2002
36.
Sandhu, H,
Wiesenthal SR,
MacDonald PE,
McCall RH,
Tchipashvili V,
Rashid S,
Satkunarajah M,
Irwin DM,
Shi ZQ,
Brubaker PL,
Wheeler MB,
Vranic M,
Efendic S,
and
Giacca A.
Glucagon-like peptide 1 increases insulin sensitivity in depancreatized dogs.
Diabetes
48:
1045-1053,
1999[Abstract].
37.
Scrocchi, LA,
Brown TJ,
Maclusky N,
Brubaker PL,
Auerbach AB,
Joyner AL,
and
Drucker DJ.
Glucose intolerance but normal satiety in mice with null mutation in the glucagon-like peptide 1 receptor gene.
Nat Med
2:
1254-1258,
1996[ISI][Medline].
38.
Trumper, A,
Trumper K,
and
Horsch D.
Mechanisms of mitogenic and anti-apoptotic signaling by glucose-dependent insulinotropic polypeptide in (INS-1)-cells.
J Endocrinol
174:
233-246,
2002
39.
Trumper, A,
Trumper K,
Trusheim H,
Arnold R,
Goke B,
and
Horsch D.
Glucose-dependent insulinotropic polypeptide is a growth factor for (INS-1) cells by pleiotropic signaling.
Mol Endocrinol
15:
1559-1570,
2001
40.
Tseng, CC,
Kieffer TJ,
Jarboe LA,
Usdin TB,
and
Wolfe MM.
Postprandial stimulation of insulin release by glucose-dependent insulinotropic polypeptide (GIP). Effect of a specific glucose-dependent insulinotropic polypeptide receptor antagonist in the rat.
J Clin Invest
98:
2440-2445,
1996
41.
Tseng, CC,
Zhang XY,
and
Wolfe MM.
Effect of GIP and GLP-1 antagonists on insulin release in the rat.
Am J Physiol Endocrinol Metab
276:
E1049-E1054,
1999
42.
Unger, RH,
and
Eisentraut AM.
Entero-insular axis.
Arch Intern Med
123:
261-266,
1969[ISI][Medline].
43.
Vaag, A,
Holst J,
Volund A,
and
Beck-Nielsen H.
Gut incretin hormones in identical twins discordant for non-insulin-dependent diabetes mellitus (NIDDM)evidence for decreased glucagon-like peptide 1 secretion during oral glucose ingestion in NIDDM twins.
Eur J Endocrinol
135:
425-432,
1996[ISI][Medline].
44.
Vilsboll, T,
Krarup T,
Madsbad S,
and
Holst J.
Defective amplification of the late phase insulin response to glucose by GIP in obese type II diabetic patients.
Diabetologia
45:
1111-1119,
2002[ISI][Medline].
45.
Ward, WK,
Beard JC,
and
Porte DJ.
Clinical aspects of islet cell function in non-insulin-dependent diabetes mellitus.
Diabetes Metab Res Rev
2:
297-312,
1986.
46.
Wheeler, MB,
Gelling RW,
McIntosh CH,
Georgiou J,
Brown JC,
and
Pederson RA.
Functional expression of the rat pancreatic islet glucose-dependent insulinotropic polypeptide receptor: ligand binding and intracellular signaling properties.
Endocrinology
136:
4629-4639,
1995[Abstract].
47.
Willms, B,
Werner J,
Holst JJ,
Orskov C,
Creutzfeldt W,
and
Nauck MA.
Gastric empying glucose responses, and insulin secretion after a liquid test mealeffects of exogenous glucagon-like peptide-1 (GLP-1)-(7-36) amide in type 2 (noninsulin dependent) diabetic patients.
J Clin Endocrinol Metab
81:
327-332,
1996[Abstract].