From the Department of Medicine, Banting and Best
Diabetes Centre, Toronto General Hospital, University of Toronto,
Ontario M5G 2C4, Canada, and the ** Louis-Jeantet Research
Laboratories, University Medical Centre,
1211 Geneva 4, Switzerland
Received for publication, September 13, 2002, and in revised form, October 22, 2002
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ABSTRACT |
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Glucagon-like peptide-1 (GLP-1) stimulates
insulin secretion and augments Glucagon-like peptide-1
(GLP-1)1 is derived from
posttranslational processing of proglucagon in enteroendocrine L cells
(1) and is secreted from the distal gut after nutrient ingestion (2). The termination of GLP-1 action by the enzyme dipeptidyl peptidase IV
occurs within minutes following GLP-1 secretion (3-5), yet GLP-1
exerts several rapid metabolic actions including stimulation and
inhibition of insulin and glucagon secretion, respectively (6-10).
GLP-1 action is essential for glucose homeostasis, because GLP-1
receptor blockade with the antagonist exendin (9-39) increases blood
glucose and decreases levels of circulating insulin in human and rodent
studies (11-14).
Activation of GLP-1 receptor signaling leads to enhanced expression of
mRNA transcripts for glucokinase, GLUT-2, Pdx-1, and insulin in GLP-1 receptor signaling is also coupled to formation of new Materials--
Tissue culture medium, serum, flasks, plates, and
antibiotics, including G418, were from Invitrogen.
Cycloheximide, forskolin, and protease inhibitor mixture were purchased
from Sigma. Exendin-4 was from California Peptide Research (Napa, CA).
Animal Experiments--
Male C57BL/6 mice, 8 weeks of age, were
used for experiments shown in Figs. 1-3. Age- and sex-matched CD-1
GLP-1R+/+ control mice housed in the same animal facility were used for
studies of GLP-1R Oral Glucose Tolerance Test and Measurement of Plasma and
Pancreatic Insulin Levels--
Oral glucose tolerance tests were
carried out after an overnight fast as described (14, 32). A blood
sample was collected from the tail vein during the 10-20 min time
period for measurement of plasma insulin using a rat insulin
enzyme-linked immunoassay kit (Crystal Chem. Inc., Chicago, IL) with
mouse insulin as a standard (14).
Histological Assessment of Islet Apoptosis and
Proliferation--
To detect apoptosis, TUNEL (terminal
deoxynucleotide transferase-mediated dUTP nick end labeling) staining
was performed using ApopTag Peroxidase in situ Apoptosis
detection kit (S7100) (Intergen Company, Purchase, NY), according to
the manufacturer's instructions as described (33). Slides were
analyzed with a Leica microscope, and apoptotic rates were
calculated as the number of TUNEL-positive cells per islet,
n = 6-7 pancreases for each experimental group of
C57Bl/6 +/+ mice, or n = 4-6 pancreases for each group
of CD1 GLP-1R+/+ or GLP-1R
Islet cell proliferation was assessed by counting the number of
5'-bromo-2'-deoxyuridine-positve (BrdUrd+) islet cells in multiple
pancreatic sections from both wild-type C57BL/6 and CD-1 and
GLP-1R Rat Islets and Sorted Quantification of Apoptosis and Cell Division in Isolated Cell Culture and Apoptosis Experiments--
BHK fibroblasts were
grown in Dulbecco's modified Eagle's medium, 4.5g/l glucose
supplemented with 5% calf serum. Cells were transfected with cDNAs
encoding the rat GLP-1 receptor cloned in the pcDNA3.1 eukaryotic
expression vector (Invitrogen, San Diego, CA). Stably transfected cell
populations were selected by growth in G418 (Invitrogen) at 0.8 mg/ml
for 2 weeks and studies of apoptosis in BHK-GLP-1R cells were done
using pools of G418-resistant clones. For apoptosis experiments, cells
were replated in culture medium lacking G418, serum-starved overnight,
and treated with cycloheximide in the presence or absence of the
indicated peptides or drugs as described (40).
Cell Viability Assay--
Cells were exposed to either vehicle
or cycloheximide in the presence or absence of the indicated drugs, and
the number of viable cells was assessed by measuring the bioreduction
of a methane thiosulforate tetrazolium salt at 490 nm using the
CellTiter 96 aqueous assay (Promega, Madison, WI.)
SDS-Polyacrylamide Gel Electrophoresis and Western Blot
Analysis--
Cell pellets were lysed at 4 °C in radioimmune
precipitation assay buffer containing a protease inhibitor mixture, and
cleared lysates were boiled in sample buffer containing
Statistical Analysis--
All values are presented as means ± S.E. Statistical significance between groups was evaluated by
student's t test or Bonferroni-corrected analysis of variance.
As treatment of rodents with GLP-1 agonists leads to increased
islet mass in association with cell mass via activation of
cell
proliferation and islet neogenesis. We examined whether GLP-1
receptor signaling modifies the cellular susceptibility to apoptosis.
Mice administered streptozotocin (STZ), an agent known to induce
cell apoptosis, exhibit sustained improvement in glycemic control and
increased levels of plasma insulin with concomitant administration of
the GLP-1 agonist exendin-4 (Ex-4). Blood glucose remained
significantly lower for weeks after cessation of exendin-4. STZ induced
cell apoptosis, which was significantly reduced by
co-administration of Ex-4. Conversely, mice with a targeted disruption
of the GLP-1 receptor gene exhibited increased
cell apoptosis after
STZ administration. Exendin-4 directly reduced cytokine-induced
apoptosis in purified rat
cells exposed to interleukin 1
, tumor
necrosis fator
, and interferon
in vitro.
Furthermore, Ex-4-treated BHK-GLP-1R cells exhibited significantly
increased cell viability, reduced caspase activity, and decreased
cleavage of
-catenin after treatment with cycloheximide in
vitro. These findings demonstrate that GLP-1 receptor signaling
directly modifies the susceptibility to apoptotic injury, and
provides a new potential mechanism linking GLP-1 receptor activation to
preservation or enhancement of
cell mass in
vivo.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
cell lines (15-17) and in both normal and diabetic rodents (18-20).
Furthermore, GLP-1 and exendin-4 promote differentiation of exocrine
cell lines toward a
cell phenotype (21), a process that appears to
depend on the expression of Pdx-1 (22, 23).
cells
through enhanced proliferation of existing
cells (24) and via
induction of islet neogenesis (25). The mitogenic actions of GLP-1 are
detectable in normal rodents (20, 24) and in the setting of
experimental diabetes (19, 25). Administration of GLP-1 or exendin-4 to
newborn rats treated with the
cell toxin streptozotocin (STZ) leads
to increased
cell mass at postnatal day 7, which persists and
remains increased at 2 months of age. The increased
cell mass in
the GLP-1/exendin-4 treated rats was attributed to both enhanced
cell proliferation and increased numbers of small budding islets (26).
Because STZ is known to induce
cell destruction in part through
activation of apoptotic pathways (27-29), we examined whether GLP-1
receptor activation influences
cell mass via regulation of cellular
susceptibility to apoptotic cell death.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
mice in the CD1 background (8-week-old male
mice). All animals were maintained on standard laboratory chow under a
12 h:12 h light-dark schedule, and experiments were conducted according to protocols and guidelines approved by the Toronto General
Hospital Animal Care Committee. STZ (Sigma) (50 mg/kg body weight,
intraperitoneal injection once daily for 5 days) was administered as a
freshly prepared solution in 0.1 mM sodium citrate pH 5.5. Exendin-4 (Ex-4; 24 nmol/kg body weight, a dose selected based on
therapeutic efficacy in previous mouse experiments (30)) was
administered as a single daily intraperitoneal injection. For studies
depicted in Figs. 1 and 2, morning blood glucose was measured
periodically throughout the experimental period and an oral glucose
tolerance tests was done at day 30. For histological studies of
islet apoptosis, Ex-4 administration was commenced either 2 or 7 days
before STZ in separate experiments and continued until the last
injection of STZ; C57BL/6 mice were sacrificed within ~24 h after the
last STZ injection. For studies of apoptosis in GLP-1R
/
mice,
wild-type GLP-1R+/+ and GLP-1R
/
mice were divided into separate
groups (n = 4-6) and administered a slightly lower
dose of STZ (40 mg/kg body weight) because of the different sensitivities of CD-1 versus C57BL/6 mice to STZ as
delineated in preliminary dose-response studies caused by the known
species-specific sensitivity to streptozotocin-induced apoptosis (31).
After completion of the experiments (~48 h after the last dose of
STZ), mice were euthanized by CO2 anesthesia, blood was
collected by cardiac puncture for plasma insulin determinations, and
pancreases were removed, fixed in 10% formalin overnight, and embedded
in paraffin for histological analyses.
/
mice. Analysis of serial consecutive
islet sections stained with either insulin or the ApopTag reagent
demonstrated that the apoptotic nuclei were localized to
insulin-immunopositive
cells.
/
CD1 mice administered BrdUrd (Roche) by intraperitoneal injection, 50 mg/kg body weight, ~ 5 h prior to removal of the pancreas. Immunohistochemical detection of BrdUrd+ cells was carried out using an anti-BrdUrd antibody (CalTag Laboratories, Burlingame, CA). Serial sections were stained for either insulin or BrdUrd, and
islet and pancreatic areas were measured using a Leica microscope and
Q500MC software. The vast majority of BrdUrd+ cells were immunopositive for insulin, and hence represented
cells. The relative
cross-sectional
cell area, as a percentage of total pancreatic
area, was assessed quantitatively as previously described (34-36). The
number of BrdUrd-positive cells are expressed both per islet and per
105 µm2
-cell area.
Cells--
Islets were isolated from
the pancreas of adult male Wistar rats (180-220 g) by collagenase
digestion and purified on a gradient of Ficoll (37). The islets were
further dissociated into single cells by trypsinization, and
cells
were sorted on the basis of their autofluorescence using a FACStar Plus
(BD Biosciences) as described (37). The sorted cell population
comprises 95%
cells (37). Cells were allowed to recover from the
isolation/sorting procedures by culture overnight in Dulbecco's
modified Eagle's medium, 10% fetal calf serum, 11.2 mM glucose using plastic dishes to which they did not
attach. For measurement of apoptosis by ELISA (see below), cells were
seeded (5-8 × 105 cells/ml, Dulbecco's modified
Eagle's medium, 11.2 mM glucose, 10% fetal calf serum, 50 µl/well) in 96-well plates pre-coated with extracellular matrix from
804G rat bladder carcinoma cells (Desmos, San Diego, CA) (38). For
TUNEL labeling (see below) and labeling with BrdUrd, the sorted
cells were seeded at the same density and in the same medium as 50-µl
microdroplets placed at the center of 35-mm-diameter plastic Petri
dishes coated with 804G matrix. This allowed for use of an
inverted-stage fluorescent microscope to examine the cells (under a
coverslip) after fixation. Sorted rat
cells maintained in monolayer
culture were exposed to a mixture of three cytokines for 18 h at
8.3 mM glucose. These conditions were established on the
basis of preliminary experiments (data not shown) designed to obtain
marked augmentation of apoptosis without significant cell necrosis or
detachment of cells from the culture vessel. All incubations of cells
were in a humidified atmosphere of 5% CO2 at 37 °C.
Exendin-4 was present throughout the 18-h incubation with or without
cytokines at a final concentration of 100 nM.
Cells--
Apoptosis of purified
cells was estimated using Cell
Death Detection ElisaPLUS (Roche Biochemicals, Mannheim,
Germany) for determination of cytoplasmic
histone-associated-DNA-fragments (mono- and oligonucleosomes) in cell
lysates, a method that correlates well with apoptosis quantification by annexin V staining (39). Alternatively, cells seeded
in microdroplets on Petri dishes were processed for estimation of
apoptosis using the TUNEL technique according to the manufacturer's instructions ("in situ cell death detection kit" from
Roche Biochemicals) following fixation for 20 min in 4%
paraformaldehyde and permeabilization using 0.5% Triton X-100 for 4 min at room temperature. Cell replication was assessed by incorporation
of BrdUrd (Sigma). For this purpose, BrdUrd (10 µM) was
included throughout the 18-h incubation with cytokines or exendin.
Cells were then fixed, and BrdUrd+ cells were visualized by immunoflueorescence.
-mercaptoethanol and stored at
70 °C. Protein concentration was
determined using bovine serum albumin as a standard and equal amounts
of cell lysates were separated by discontinuous SDS-polyacrylamide gel
electrophoresis under reducing conditions and electrotransferred onto
Hybond-C nitrocellulose membrane (Amersham Biosciences). The resultant blot was blocked with 5% skim milk in phosphate-buffered saline containing 0.2% Tween 20 and incubated with the indicated primary antibody overnight at room temperature. Proteins were detected with a
secondary antibody conjugated to horseradish peroxidase and an enhanced
chemiluminescence commercial kit (Amersham Biosciences). Western blot
analyses were carried out using primary antibodies reactive to active
caspase-3 p17 subunit (1:1000 dilution; PerkinElmer Life Sciences),
cytochrome c (1:250 dilution; BIOSOURCE
International), porin/VDAC 31HL (1:500 dilution; Calbiochem), Akt
(1:1000; Cell Signaling Technology (Beverly, MA)) and actin (1:5,000
dilution; Sigma).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
cell proliferation and islet neogenesis (19, 25), we hypothesized that GLP-1 might also enhance
cell mass via protection from cellular apoptosis. To test this
hypothesis, wild-type C57BL/6 mice were treated with low-dose
streptozotocin, a chemical known to induce
-cell apoptosis (29), in
the presence or absence of the GLP-1 analog Ex-4, administered for 2 days before STZ, during, and 3 days after STZ (Fig.
1). The pretreatment regimen was selected
in part because of observations that pretreatment of mice with the
related glucagon-like peptide GLP-2 significantly reduced apoptosis
in experimental models of intestinal injury (33, 41). Mice treated with
STZ developed progressive hyperglycemia, with levels of blood glucose
rising steadily several days after STZ administration. In contrast,
mice that received both STZ (5 days) and Ex-4 (10 days) exhibited a significantly delayed onset of hyperglycemia (compare day 9-12 glucose
in STZ versus STZ+Ex-4 mice, Fig. 1) and blood glucose remained significantly lower even 2 weeks after the last dose of Ex-4
(Fig. 1, p < 0.05 for STZ alone versus
STZ+Ex-4 glucose between day 9-29). Furthermore, levels of circulating
insulin at day 30 were significantly greater in STZ+Ex-4 mice, 20 days after the last Ex-4 injection (Fig 1).
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Fig. 1.
Morning-fed blood glucose and plasma insulin
in wild-type C57BL/6 mice treated with saline, exendin-4 (Ex-4), STZ,
or STZ+Ex-4. Blood glucose was significantly lower from day 9 to
day 29 in STZ+Ex-4 mice compared with glucose in mice receiving STZ
alone (p < 0.05), whereas fed plasma insulin level
measured at day 30 was significantly higher in STZ+Ex-4 mice
versus STZ alone (*, p < 0.05);
n = 10 mice per each experimental group. C,
control (saline).
A separate experiment was carried out using a different pre-treatment
period starting exendin-4 administration 7 days before STZ, and
continuing exendin-4 administration for a total of 28 days, with
assessment of oral glucose tolerance and glucose-stimulated insulin at
day 30. A similar protective response to Ex-4 was observed in this
longer experiment shown in Fig 2.
Although hyperglycemia developed in all mice treated with STZ, levels
of blood glucose were significantly lower in the STZ+Ex-4 group, even
more than 3 weeks after cessation of Ex-4 (Fig. 2a;
p < 0.05 for day 15-52 glucose, STZ versus
STZ+Ex-4). Oral glucose tolerance testing on day 30, 2 days after the
last dose of Ex-4, revealed significantly lower glucose excursion
specifically at early time points following oral glucose loading, in
association with significantly increased levels of plasma insulin in
STZ+Ex-4 mice (Fig. 2b; p < 0.002). Furthermore, the levels of fed plasma insulin remained significantly greater in the STZ+Ex-4-treated mice and were comparable to levels detected in Ex-4-alone mice that did not received streptozotocin (p < 0.05) at day 55, 27 days after the last dose of
exendin-4 (Fig. 2a).
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To ascertain the mechanisms underlying the sustained improvement in
levels of glucose and insulin in STZ+Ex-4 mice, we assessed pancreatic
histological sections for the presence of apoptotic cells in
separate groups of mice treated with STZ, with or without Ex-4. Only a
rare apoptotic
cell was detectable in histological sections from
pancreases of control or Ex-4-treated mice in the absence of STZ (Fig
3a). In contrast,
morphological features of apoptosis, including pyknotic nuclei, were
readily detectable in pancreatic sections from STZ-treated mice. The
numbers of TUNEL-positive apoptotic
cells were markedly increased
in STZ-treated mice, and significantly reduced (4.5-fold) in mice
administered both STZ and Ex-4, whether expressed as the number of
apoptotic cells per islet or normalized to relative
cell area (Fig.
3, b and d; p < 0.001, STZ
versus STZ+Ex-4).
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Because GLP-1 agonists have been shown to induce -cell proliferation
(19), we assessed the extent of
-cell proliferation and expansion of
islet mass in the same experiment. Wild-type mice treated with Ex-4
alone for 7 days in the absence of STZ exhibited a greater than 2-fold
increase in the number of BrdUrd+
cells, whether expressed as
BrdUrd+ cells per islet, or normalized to
cell area (Fig. 3,
c and e, respectively; p < 0.05 for control versus Ex-4-treated mice). In contrast, we did
not detect a significant increase in the number of BrdUrd+ cells in
Ex-4-treated mice treated for 5 consecutive days with STZ (Fig. 3,
c and e).
These findings demonstrate that exogenous activation of GLP-1 receptor
signaling reduced STZ-associated islet apoptosis in wild-type mice
in vivo. To ascertain whether basal levels of endogenous GLP-1 receptor signaling protected cells from external injury, we
administered STZ to mice with a targeted disruption of the Glp-1R gene (GLP-1R
/
mice (32)). Blood glucose
increased more rapidly in GLP-1R
/
versus GLP-1R+/+ mice
after STZ administration (Fig. 4,
c and e; p < .05 for glucose at
day 7 in STZ-treated GLP-1R+/+ versus GLP-1R
/
mice) and
remained significantly greater in STZ-treated GLP-1R
/
from day
7-16 (Fig. 4, c and e; p < 0.05; GLP-1R
/
versus control GLP-1R+/+ mice treated with
STZ). In contrast, after day 16, the levels of blood glucose in
STZ-treated mice remained elevated however no significant differences
in glucose (Fig. 4, c) were detected in GLP-1R
/
versus GLP-1R+/+ by day 28, 23 days after the last dose of
STZ. Similarly, levels of glucose-stimulated insulin at day 7 were not
significantly different. In contrast, the number of apoptotic
cells
detected ~48 h after the last dose of STZ was increased in both
GLP-1R+/+ and GLP-1R
/
mice and was significantly greater (2.7-fold)
in GLP-1R
/
mice treated with identical doses of STZ (Fig. 4,
a and b; p < 0.002). Taken together, the data presented in Figs. 1-4 demonstrate that activation or abrogation of GLP-1 receptor signaling regulates the extent of
murine
cell apoptosis in vivo.
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To determine whether GLP-1 agonists exert direct anti-apoptotic effects
on islet cells in vitro using a different approach for
generation of cytotoxic injury, we induced apoptosis in purified populations of sorted rat
cells using a combination of cytokines (1 ng/ml interleukin 1
, 5 ng/ml tumor necrosis factor
, 5 ng/ml interferon
) as previously described (42). Incubation with cytokines
alone for 18 h produced a 4.9-fold increase in apoptosis, however
co-incubation with cytokines and exendin-4 significantly reduced the
extent of apoptosis (Fig 5a)
by 44.0 ± 5.2% compared with the extent of apoptosis with
cytokines alone (p < 0.001). To assess directly the
percentage of apoptotic cells under these various conditions,
individual rat
cells were examined for TUNEL staining (Fig.
5b). Cytokines increased the percentage of TUNEL-positive
cells from 0.6 to 4.2%. Consistent with results seen by ELISA, exendin-4 significantly reduced the percentage of TUNEL-positive
cells compared with values seen with cytokines alone (Fig.
5b; p < 0.001). Because GLP-1 agonists are
known to stimulate
cell proliferation, it was possible that the
observed quantitative effects on apoptosis were to some degree modified
by increased cell replication. To test this possibility, cell division
was estimated by incorporation of BrdUrd (present throughout the
18 h incubation period). As expected for primary adult
cells,
the number of dividing cells was extremely low under control
conditions. Although there did appear to be a modest increase with
exendin-4 treatment, no more than 10 BrdUrd-fluorescent cells were
visible in each monolayer culture of 25,000 cells (amounting to less
than 0.05%) even under these conditions. In the presence of cytokines, the cells change their morphology making it difficult to observe BrdUrd-positive cells with any accuracy, but there was no evidence for
any increase in cell division. Similarly, glucose-stimulated insulin
secretion was abolished by treatment with the cytokines and exendin-4
was not able to reverse this impairment (data not shown), in keeping
with the known and severe effects of this combination of cytokines on
-cell function (43).
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These findings demonstrate that direct activation of the -cell GLP-1
receptor is coupled to reduction of apoptosis in wild-type mice
in vivo and in primary rat
-cell cultures in
vitro. To determine whether GLP-1 receptor activation is
sufficient for direct engagement of antiapoptotic pathways in
heterologous cells, we introduced the rat GLP-1 receptor into BHK
fibroblasts. BHK-GLP-1R cells responded to GLP-1 with a
dose-dependent increase in cAMP accumulation, whereas GLP-1
had no effect on wild-type BHK cells (data not shown). After treatment
with cycloheximide, BHK cells exhibit morphological features associated
with apoptosis including membrane blebbing, cell shrinkage and
detachment, and cell fragmentation into apoptotic bodies (40).
BHK-GLP-1R cells treated with 80 µM cycloheximide exhibited progressively reduced cell viability, whereas co-incubation of cells with Ex-4 significantly increased cell viability at multiple time points (Fig. 6a). The
relative activities of caspase-3, caspase-8, and caspase-9 were
markedly induced after exposure to cycloheximide and significantly
reduced after treatment with Ex-4 in the same experiments (data not
shown). Cycloheximide-treated BHK-GLP-1R cells exhibited increased
levels of the active p17 caspase 3 subunit that were markedly
diminished by treatment with Ex-4 (Fig. 6b). Similarly, Ex-4
reduced the translocation of cytochrome c from the
mitochondrial compartment to the cytosol in the setting of cycloheximide-induced apoptosis (Fig. 6c). Furthermore, the
levels of intact
-catenin, a caspase-3 substrate, and the
prosurvival kinase Akt, were markedly reduced following exposure to
cycloheximide, whereas Ex-4 treatment clearly preserved levels of both
intact
-catenin and Akt (Fig. 6d).
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DISCUSSION |
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Original concepts of GLP-1 action focused on its role as a
gut-derived incretin acting on islet cells to augment
nutrient-stimulated insulin release in the postprandial state (44, 45).
The finding that GLP-1 agonists also promote
cell proliferation and
islet neogenesis (19, 25), taken together with the defects in islet topography observed in the GLP-1R
/
mouse (35) has broadened the
physiological actions of incretin peptides to encompass the regulation
of
cell mass. Indeed, recent evidence suggests a potential role for
the structurally related incretin, glucose-dependent insulinotropic polypeptide as a second gut-derived regulator of
-cell proliferation (46).
The concept that circulating gut peptides exert cytoprotective actions on distal target tissues is exemplified by the actions of GLP-2, a proglucagan-derived peptide co-secreted from the L cell together with GLP-1 (47, 48). GLP-2 promotes cell proliferation indirectly via activation of a distinct G-protein-coupled receptor expressed in human enteroendocrine cells and murine enteric neurons, leading to expansion of the mucosal epithelium in the small and large bowel (49-53). Although the anti-apoptotic actions of GLP-2 are not readily apparent in the normal epithelial mucosa, induction of experimental intestinal injury is associated with increased crypt apoptosis that is markedly suppressed by exogenous GLP-2 administration (33, 41).
Given the low basal rate of apoptosis in the normal islet (54),
induction of islet injury is necessary to unmask the anti-apoptotic effects of GLP-1 on the cell in vivo. Rats treated with
intraperitoneal STZ for 5 days exhibited increased pancreatic levels of
prohormone convertase-1, the primary enzyme responsible for liberation
of GLP-1 from proglucagon (55). Furthermore, the levels of bioactive GLP-1(7-36 amide) were significantly elevated in the circulation and
pancreas of STZ-treated diabetic rats (55). These findings suggest that
at least some types of experimental diabetes associated with
-cell
injury may be characterized by enhanced liberation of both pancreatic
and intestinal GLP-1, which may have physiological implications for
protection of vulnerable
cells from further cell death.
A recent report suggests that GLP-1 may exert cytoprotective effects in
rat islets incubated with cytokines in vitro (56). Because
primary cultures of rat islets generally contain a mixture of both
islet and contaminating non-islet cell types (57), it is not possible
to determine whether the antiapoptotic effects of GLP-1 in mixed islet
cultures are exerted through direct or indirect actions on the islet
cell. Furthermore, the GLP-1 receptor has been localized to not
only
but also to islet
and
cells (58); hence even mixed
islet endocrine cultures free of exocrine contamination will contain
several distinct cell types capable of responding to exogenous GLP-1
in vitro.
To determine whether GLP-1 exerts antiapoptotic effects via direct or
indirect mechanisms, we used two independent experimental models,
highly purified sorted rat cells and heterologous cells transfected
with the GLP-1 receptor. The finding that GLP-1 directly inhibits
apoptosis both in populations of islet
cells exposed to cytokines
and in BHK-GLP-1R fibroblasts treated with cycloheximide strongly
implicates a direct antiapoptotic effect of GLP-1 agonists transduced
via the GLP-1 receptor. Similarly, heterologous cells stably
transfected with the GLP-2 receptor exhibit enhanced survival after CHX
treatment and reduced activation of proapoptotic effector caspases
after treatment of cells with GLP-2 (33, 40). Because both GLP-1 and
GLP-2 increase the levels of cAMP, and forskolin mimics the
antiapoptotic actions of these peptides in vitro, activation of downstream cAMP-dependent prosurvival pathways may be an
important feature of several G-protein-coupled receptors that regulate
cellular apoptosis. Indeed, the vasoactive intestinal peptide
and pituitary adenylate cyclose-activating peptide receptors
are also expressed on islet
cells (59) and coupled to adenylate
cyclase activation and these insulinotropic peptides exert
antiapoptotic and cytoprotective actions in vitro (60-63).
Nevertheless, elevated levels of intracellular cAMP are not always
associated with enhanced
-cell survival (64), hence the precise
signal-transduction mechanisms linking G-protein-coupled receptor
activation to
-cell injury require further clarification.
The majority of actions ascribed to GLP-1 have been deduced following
exogenous GLP-1 administration and some, but not all of these effects
are physiologically important for metabolic regulation and -cell
function. GLP-1R
/
cells exhibit reduced levels of cAMP, defects
in glucose-stimulated insulin secretion (32), and abnormalities in
glucose-stimulated calcium signaling (65). Nevertheless,
ob/ob:GLP-1R
/
mice exhibit enhanced islet proliferation and
up-regulation of islet mass despite the complete absence of GLP-1R
signaling (34). In contrast, the finding that GLP-1R
/
cells
exhibit enhanced susceptibility to STZ-induced apoptosis demonstrates
that GLP-1 receptor signaling is an important physiological determinant
of
-cell survival following external injury. Because GLP-1 analogues
are currently being developed for the treatment of type 2 diabetes, a
disease characterized by progressive deterioration and ultimate loss of
cell function, understanding the cytoprotective and proliferative
mechanisms activated by GLP-1 in the islet
cell is potentially
relevant to the therapy of type 2 diabetes.
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FOOTNOTES |
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* This work was partially supported by grants from the Juvenile Diabetes Research Foundation (JDRF 2000-559 to D. J. D. and JDRF 4-1999-844 to P. A. H.), the Canadian Diabetes Association and Ontario Research and Development Challenge Fund (to D. J. D.), and by the Swiss National Science Foundation (Grant 3200-061776.00 to P. A. H.).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.
§ These authors contributed equally to this work.
¶ Supported by a postdoctoral fellowship Award from the Canadian Diabetes Association.
Supported by a Novo Nordisk-Banting and Best Diabetes Centre studentship.
Supported by a Senior Scientist Award (Canadian Institutes of
Health Research). To whom correspondence should be addressed: Toronto General Hospital, 200 Elizabeth St. MBRW4R-402, Toronto, Ontario M5G 2C4, Canada. Tel.:416-340-4125; Fax: 416-978-4108; E-mail: d.drucker@utoronto.ca.
Published, JBC Papers in Press, October 29, 2002, DOI 10.1074/jbc.M209423200
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ABBREVIATIONS |
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The abbreviations used are: GLP-1, glucagon-like peptide-1; STZ, streptozotocin; Ex-4, exendin-4; TUNEL, terminal deoxynucleotide transferase-mediated dUTP nick end labeling; BrdUrd, 5'-bromo-2'-deoxyuridine; ELISA, enzyme-linked immunosorbent assay; BHK, baby hamster kidney; CHX, cycloheximide.
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