Mutations to the Third Cytoplasmic Domain of the Glucagon-Like Peptide 1 (GLP-1) Receptor Can Functionally Uncouple GLP-1-Stimulated Insulin Secretion in HIT-T15 Cells
Anne Marie F. Salapatek,
Patrick E. MacDonald,
Herbert Y. Gaisano and
Michael B. Wheeler
Departments of Medicine and Physiology University of
Toronto Toronto, Ontario, Canada M5S 1A8
 |
ABSTRACT
|
---|
Glucagon-like peptide-1 (GLP-1) is an
insulinotropic hormone with powerful antidiabetogenic effects that are
thought to be mediated by adenylyl cyclase (AC). Recently, we generated
two GLP-1 receptor mutant isoforms (IC31 and DM-1) that
displayed efficient ligand binding and the ability to promote
Ca2+ mobilization from intracellular stores but
lacked the ability to couple to AC. In the present study, the wild-type
rat GLP-1 receptor (WT-GLP-1 R) or the IC31
and DM-1 mutant forms were expressed for the first time in the
insulin-producing HIT-T15 cells. Only cells expressing
WT-GLP-1 R displayed dramatically elevated
GLP-1-induced cAMP responses and elevated insulin
secretion. The increase in GLP-1-stimulated secretion in
cells expressing WT-GLP-1 R, however, was not accompanied
by differences in glucose-stimulated insulin release. Prolonged
exposure to GLP-1 (10 nM, 17
h), not only led to an increase in insulin secretion but also increased
insulin mRNA levels, but only in cells expressing the WT-GLP-1R and not
the mutant isoforms. Electrophysiological analyses revealed that
GLP-1 application enhanced L-type voltage-dependent
Ca2+ channel (VDCC) currents > 2-fold and
caused a positive shift in VDCC voltage-dependent inactivation in
WT-GLP-1R cells only, not control or mutant (DM-1) cells. This action
on the Ca2+ current was further enhanced by the
VDCC agonist, BAYK8644, suggesting GLP-1 acts via a
distinct mechanism dependent on cAMP. These studies demonstrate that
the GLP-1 receptor efficiently couples to AC to stimulate
insulin secretion and that receptors lacking critical residues in the
proximal region of the third intracellular loop can effectively
uncouple the receptor from cAMP production, VDCC activity,
insulin secretion, and insulin biosynthesis.
 |
INTRODUCTION
|
---|
The incretin hormone glucagon-like peptide-1 (GLP-1)
is released from intestinal L cells to stimulate insulin secretion from
pancreatic ß-cells in a glucose-sensitive manner. The biologically
active forms of GLP-1, GLP-1(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37), and
GLP-1(736 amide), have been shown to be among the most
potent insulinotropic agents identified to date in mammals (1).
Clinical studies indicate that GLP-1 not only stimulates
insulin secretion in normal subjects, but also in those with
non-insulin-dependent diabetes mellitus (type 2) (2, 3, 4), supporting its
therapeutic potential. A possible role for GLP-1 in the
central control of feeding has also been demonstrated (5).
GLP-1 administered via an
intracerebroventricular injection was a powerful inhibitor
of feeding in fasted rats.
The insulinotropic properties of GLP-1 are mediated
through a high-affinity GLP-1 receptor on the
insulin-secreting ß-cells of the pancreas (6, 7). The receptor cDNA,
initially cloned from rat pancreatic islet cells, and subsequently from
human pancreas (8, 9), predicts a seven-transmembrane G protein-coupled
receptor (GPCR) of the glucagon/vasoactive intestinal
peptide/secretin receptor subfamily (10). Work on the endogenous
receptor in isolated ß-cells and ß-cell lines, and with the
recombinant GLP-1 receptors expressed in cell lines,
strongly suggests that the insulinotropic actions of GLP-1
are mediated by cAMP-dependent activation of protein kinase A (11). The
mechanisms whereby protein kinase A (PKA) mediates
GLP-1-induced insulin exocytosis appears to be
multifaceted including proposed actions on ATP-sensitive K+
channels, nonselective cation channels, L-type voltage-dependent
Ca2+ channels (VDCCs), and on the exocytotic machinery
(12, 13, 14, 15, 16).
The majority of studies correlating GLP-1-induced insulin
secretion with PKA have relied heavily on the use of cAMP analogs with
varying degrees of specificity for the activity of this kinase. In the
present study, we have used a novel approach to correlate the
insulinotropic actions of GLP-1 with cAMP, employing a
series of recombinant GLP-1 receptor isoforms that are
specifically uncoupled from adenylyl cyclase (AC). Previously, we and
others reported that the third intracellular loop (IC3) and the
carboxyl-terminal (CT) domain were shown to contain specific amino
acids required for efficient signaling of the receptor (17, 18, 19, 20). Two
adjacent amino acid block deletion mutations in the predicted
N-terminal portion of the IC3 domain (DM-1, lacking V331-I332-A333, and
IC31, lacking K334-L335-K336) were shown to be required for the
efficient coupling of receptor to AC (18, 19) when expressed in COS
cells. In the present study these mutant receptor isoforms and the
recombinant WT-GLP-1R have been examined functionally in
the insulin-producing ß-cell line HIT-T15.
 |
RESULTS
|
---|
GLP-1R Expression
To examine the level of expression of endogenous
GLP-1Rs in HIT-T15 cells and to assess the level of
expression of the wild-type (WT) GLP-1R and mutant
isoforms, Northern blotting and standard competitive binding
displacement assays were performed (Fig. 1
and Table 1
). Using the rat receptor as a probe,
under moderate stringency, GLP-1R transcripts were not
observed in HIT cells (data not shown); however, specific binding was
detected, albeit at extremely low levels (Fig. 1
, A and B, and Table 1
). Cells transfected with pCDNA-3 ß-gal demonstrated high-level
transient transfection efficiency (4580%, data not shown).
Transfection also resulted in the efficient expression of receptor
cDNAs as determined by Northern blotting (data not shown). Binding
assays revealed that transfection resulted in the functional expression
of the receptor cDNAs with specific binding (Bmax) of the
WT-GLP-1R, IC31, and DM-1 (3049 ± 478, 3347
± 530, 2939 ± 843 cpm/106 cells, respectively) found
to be similar. The affinity for binding (IC50) was not
found to differ significantly among the receptor isoforms (WT-GLP-1R,
6.5 ± 1.7 nM; IC31, 4.6 ± 2.0 nM;
and DM-1, 3.4 ± 1.8 nM, n
6). These data are
consistent with the Bmax and IC50 values
reported for these receptor isoforms expressed in COS-7 cells (18, 19).

View larger version (24K):
[in this window]
[in a new window]
|
Figure 1. Expression of GLP-1R Constructs in
HIT-T15 Cells
Binding displacement of 125I-GLP-1 in control
HIT-T15 cells and cells expressing the GLP-1R isoforms. Data are
expressed as specific binding in counts per min (A) and percentage
B/Bo (B).
|
|
Analysis of GLP-1-Stimulated Insulin Secretion
To analyze the effects of GLP-1 in HIT-T15
cells overexpressing the receptor isoforms, cells were prepared as
described in Materials and Methods. The patterns of basal
cAMP production (5 mM glucose with no peptide) and insulin
secretion were not significantly different among the test groups (Fig. 2
, A and B). GLP-1 (10
nM), in the presence of 5 mM glucose, elicited
a large cAMP response (Fig. 2C
) in cells expressing WT GLP compared
with control cells (13.9 ± 2.4 vs. 4.6 ± 1.0
pmol/well, respectively), which was accompanied by a significant
increase in insulin secretion compared with control cells (60 ± 9
vs. 30 ± 7 ng/well/h, Fig. 2D
). Increases in cAMP
accumulation and insulin secretion in response to GLP-1
were considered significant compared with control cells and those
expressing the mutant receptor isoforms (n
7; P
0.001 and P
0.01 for cAMP and insulin secretion,
respectively). Collectively, these studies suggest a strong correlation
between cAMP accumulation induced by GLP-1 and insulin
secretion. They also demonstrated that the receptor can be functionally
uncoupled from cAMP and insulin secretion in a ß-cell line through a
modification to the proximal portion of the third intracellular
loop.

View larger version (26K):
[in this window]
[in a new window]
|
Figure 2. Basal and GLP-1-Stimulated cAMP
Accumulation and Insulin Secretion in HIT-T15 Cells
HIT-T15 cells transfected with WT-GLP-1R or receptor
mutants were analyzed for total cAMP content and insulin secretion rate
under basal conditions (5 mM) glucose (A and B) and in the
presence of 10 nM GLP-1 (C and D) over a 2-h
test period.
|
|
Effects on Glucose-Stimulated Insulin Secretion
GLP-1R overexpression has been shown to
increase glucose responsivity in RIN cells in the absence of a
GLP-1 stimulus (21). To examine this possibility in
HIT-T15 cells, those expressing the WT-GLP-1R were
examined for responsiveness to glucose and compared with control cells
and those expressing the cAMP-defective mutant isoforms. Basal (0
mM glucose) insulin secretion (Fig. 3
) was not found to differ among control,
WT-GLP-1R, IC31, and DM-1 transfection groups (15.2
± 2.0, 14.0 ± 1.2, 15.3 ± 1.1, and 13.0 ± 2.1
ng/well/h, respectively; P > 0.05, n
7). Ten
millimolar glucose elicited approximately a 2-fold increase in insulin
secretion; however, no significant difference was observed among
control, WT-GLP-1R, IC31, and DM-1 transfectants
(36.3 ± 3.0, 37.3 ± 3.9, 39.5 ± 3.9, and 38.6 ±
4.9 ng/well, respectively; P > 0.05, n
7).

View larger version (23K):
[in this window]
[in a new window]
|
Figure 3. Comparison of WT-GLP-1R with Deletion
Mutations on Glucose-Stimulated Insulin Secretion
Cells in each test group were treated in the absence or presence of 10
mM glucose and insulin measured over a 2-h test period.
|
|
Effects on Insulin Biosynthesis
In COS-7 cells expressing DM-1 or IC31, receptor coupling to AC
was dramatically reduced (18, 19). To examine the effects of receptor
isoform overexpression on cAMP accumulation and insulin-secretory
function in insulin-secreting cells, the HIT-T15 cell line was
transfected with either expression vector, WT-GLP-1R, or
DM-1. For these studies, cAMP content, insulin secretion rate, total
insulin content, and insulin mRNA abundance were examined under basal
conditions and in the presence of GLP-1 (10 nM
or 1 µM) during a 17-h treatment period. As shown in Fig. 4A
, under basal conditions, cAMP content
was not significantly different among control cells and the test groups
(control, 5.4 ± 0.4 pmol/well; WT-GLP-1R, 6.1
± 1.0 pmol/well; and DM-1, 6.6 ± 1.1 pmol/well; n
6,
P > 0.77). Insulin secretion rate over the 17-h period
(Fig. 4B
) in cells expressing control, WT-GLP-1R, or DM-1
were also not found to differ significantly (23 ± 3, 28 ±
3, and 22 ± 4 ng/well/h, respectively; n = 8). Total
cellular insulin content (Fig. 4C
) was also similar in control and
WT-GLP-1R and DM-1 transfected cells (1618 ± 371,
1454 ± 300, and 1601 ± 327 ng/well, respectively; n =
8, P > 0.9). GLP-1 treatment increased
cAMP accumulation and insulin secretion maximally at 100
nM; however, insulin content was not affected by exposure
to GLP-1 for the 17-h test period (Fig. 4
, AC).
GLP-1 failed to significantly change these parameters in
control and DM-1-transfected cells. Northern blot analysis and
subsequent quantification (insulin/18 s, Fig. 4D
) revealed a
significant increase in proinsulin gene transcripts in cells expressing
WT-GLP-R at the 10 nM GLP-1 concentration
(n = 3, P = 0.05), with similar levels being
observed in control cells and those expressing the DM-1 mutant. No
significant differences in transcript levels were observed under basal
conditions or in the presence of 1 µM
GLP-1 (Fig. 4D
). These studies appear to indicate that
GLP-1 has more profound effects on insulin secretion than
on biosynthesis.

View larger version (25K):
[in this window]
[in a new window]
|
Figure 4. Comparison of WT-GLP-1R with Deletion
Mutations on Insulin Biosynthesis under Basal Conditions and in the
Presence of GLP-1 over a 17-h Test Period
cAMP accumulation in the presence of 0, 10 nM, and 1
µM GLP-1 (panel A), insulin secretion (panel
B), total cellular insulin content (panel C), and insulin mRNA
abundance as presented in relative density units (R.D.U.) (panel
D).
|
|
Effects on L-Type VDCC Currents
Cells expressing the WT-GLP-1R or DM-1 receptors tagged with
enhanced green fluorescent protein (EGFP) were shown to bind
GLP-1. Only cells expressing WT-GLP-1R showed a
GLP-1-mediated increase in cAMP (data not shown) similar
to cells expressing WT-GLP-1R. Specific HIT cells expressing the
receptors or control cells (cells expressing EGFP only), were
identified under UV light (480 nm) (typical GFP-positive cells shown in
Fig. 5
). When studied under current clamp
conditions with standard high K+ pipette solution, the mean
resting membrane potentials were not significantly different for that
recorded for control compared with WT-GLP-1R-expressing cells,
-61.4 ± 2.2 mV vs. -59.3 ± 1.5 mV. To enhance
VDCC current amplitude and reduce the rate of VDCC run-down,
Ba2+ was used as the charge carrier and resulted in a mean
peak steady state Ba2+ current of -206 ± 54 pA at
+10 mV (Fig. 6B
, control). Nifedipine (10
µM) or Cd2+ (50 µM) markedly
suppressed the inward current (not shown). Taken together, these
results indicate that the current was carried by Ba2+
through L-type VDCCs. There was no difference in VDCC current magnitude
under basal, unstimulated conditions among WT-GLP-1R, DM-1, or control
cells (Fig. 6
). As shown in Fig. 6
, B and C, GLP-1
(10-8 M) caused a leftward shift in the
current-voltage curve with a marked increase in peak VDCC current
magnitude over the -30 to +30 mV range in WT-GLP-1R compared with
control cells (-487 ± 68 pA and -201 ± 51 pA at +10 mV,
respectively; P < 0.001, n = 31 cells).
GLP-1 had no effect on DM-1 cells or control cells (Fig. 6C
). These effects on VDCC current magnitude and voltage dependence are
consistent with the positive shift in the steady state inactivation
curve observed after GLP-1 (10-8
M) application in WT-GLP-1R cells (Fig. 7
). This shift was expressed by
parameters calculated from the Boltzmann function (as described in
Materials and Methods) in which there was a significant
shift in the half-maximal voltage of inactivation (V1/2)
and the slope factor (k) in the basal (untreated) state compared
with treatment with GLP-1 (V1/2: -26.45
± 2.5 mV and -16.29 ± 4.3 mV; and k: 6.6 ± 0.4 mV and
5.2 ± 0.7 mV, respectively; P < 0.01, n =
16 cells). To gain insight into the mechanism of action of
GLP-1 on VDCC, the combinatory effects of the VDCC agonist
BAYK8644 (BAYK) and GLP-1 were tested (Fig. 8
). BAYK (10 µM), added
alone, increased VDCC current to the same extent in
WT-GLP-1R, DM-1, and control cells (an additional increase
in VDCC current of 56.4 ± 7.1%, 73.5 ± 11.3%, 64.1
± 13.5% over basal levels, respectively; see Fig. 8C
;
P < 0.01, n = 15 ). After pretreatment with BAYK
(10 µM), GLP-1 (10-8)
application further increased VDCC currents in WT-GLP-1R
cells only (an additional 62.3 ± 17.2% over BAYK addition;
P < 0.01 n = 12 cells; see Fig. 8C
). After
pretreatment with GLP-1 (10-8), BAYK (10
µM) addition caused a further increase in VDCC current in
WT-GLP-1R cells only (an additional 29.8 ± 9.9%
over GLP-1 addition, P < 0.01 or by a
greater than 2-fold increase (171.1 ± 8.5%) over BAYK addition
alone; P < 0.01, n = 10 cells; see
Fig. 8B).

View larger version (87K):
[in this window]
[in a new window]
|
Figure 5. HIT-T15 Cells Expressing WT-GLP-1R-GFP
Cells transfected with WT-GLP-1R-GFP are viewed under
standard light conditions (A) or under a UV light source (480 nm) (B)
approximately 48 h posttransfection.
|
|

View larger version (24K):
[in this window]
[in a new window]
|
Figure 6. Effect of GLP-1 on VDCCs in
GLP-1R-Expressing Cells
A, Representative sequential VDCC current traces from a single
WT-GLP-1 R cell in which one voltage step protocol was
applied in which cells are held at -70 mV and stepped up to +10 mV.
VDCC current is increased significantly with 10-8
M GLP-1 addition. B, Current-voltage curve for
WT-GLP-1R cells in the basal, unstimulated state
(open circles) and after application of
GLP-1 (10-8 M). C, Mean peak
steady state VDCC current measured in the presence of increasing
GLP-1 doses in control (open bars), DM-1
(solid bars), and WT-GLP-1R (hatched
bars) cells. VDCC current was significantly altered by
GLP-1 (10-8 M) in
WT-GLP-1R cells alone.
|
|

View larger version (16K):
[in this window]
[in a new window]
|
Figure 7. Effect of GLP-1 on WT-GLP-1R VDCC
Inactivation
A, Using a standard double-pulse protocol pictured at the top,
representative sequential VDCC traces are shown obtained from a single
WT-GLP-1R cell under basal and GLP-stimulated
(10-8 M) conditions. B, Mean steady state
inactivation curves for VDCC current obtained under basal (open
circles) and GLP (10-8 M)-stimulated
(filled circles) conditions. The fitted curves are
obtained from the Boltzmann equation as described in the text.
|
|

View larger version (25K):
[in this window]
[in a new window]
|
Figure 8. Effects of GLP-1 and BAYK8644 on VDCC
Currents
A, Superimposed VDCC current traces from one cell obtained using a
one-step voltage protocol, illustrating the sequential, cumulative
responses in the basal state (labeled 1), after GLP-1
(10-8 M) (labeled 2) and after BAYK (labeled
3) in control, WT-GLP-1R, and DM-1 cells. B, Mean peak
steady state VDCC currents measured in the basal state (open
bars) and after the sequential, cumulative addition of
GLP-1 (10-8 M) (hatched
bars) and then BAYK (solid bars). C, Mean peak
steady state VDCC currents measured in the basal state (open
bars) and after the sequential, cumulative addition of BAYK
(solid bars) and then GLP-1
(10-8 M) (hatched bars).
|
|
 |
DISCUSSION
|
---|
It is postulated that G protein activation results from
the formation of a high-affinity complex between a GPCR, its ligand,
and the heterotrimeric G protein. Studies involving the structural
alteration of two closely related members of the B class of GPCRs
(GLP-1R and the glucagon receptor) provide strong evidence
that the cytoplasmic (IC) domains of these receptors facilitate G
protein activation. Chicchi et al. (22) using a series of
deletion mutations to the glucagon receptor showed complete loss of
coupling to AC with a receptor isoform lacking eight residues in IC2
domain and dramatically reduced coupling with a series of deletions
directed to the IC3 domain. Employing a similar strategy to the IC2
loop of the GLP-1R, we also described decreases in
GLP-1-activated cAMP production but attributed this
attenuation to reduced functional receptor expression. Heller et
al. (20) reported that a point mutation to R348G compromised AC
activation by GLP-1, supporting a role for the IC3 loop in
G protein coupling within the GLP-1R. We have also
characterized the IC3 loop of the GLP-1R, between residues
K334-K351, using a series of deletion and substitution mutations
scanning the region (18, 19). As a logical extension of these studies,
we have now examined the functional consequences of this uncoupling on
GLP-1-ß-cell signal transduction.
In COS cells expressing DM-1 or IC31, receptor coupling to AC was
dramatically reduced compared with cells expressing
WT-GLP-1R, while basal production was not affected (18, 19). In HIT-T15 cells transfected with WT-GLP-1R, DM-1, or
IC31, basal cAMP content was also not significantly different (Fig. 2A
) nor were the levels of basal insulin secretion (Fig. 2B
). These
studies were repeated in growth (Fig. 4A
) or serum-free media (data not
shown) with similar negative results, suggesting that a biologically
active GLP-1 component of the serum is negligible. The
similarity in cAMP levels over 2- and 17-h test periods (Figs. 2
and 4
)
in the absence of exogenous GLP-1 also suggests that
HIT-T15 cells are not producing significant amounts of biologically
active GLP-1. Interestingly, insulin-producing RIN
104638 cells stably expressing the rat WT-GLP-1
displayed elevated basal cAMP levels and insulin secretion, elevations
that were evident in the absence and presence of glucose in the
incubation medium (21). The authors suggest several possibilities for
the elevated responses, including the possibility that the receptor is
constitutively active when overexpressed in RIN cells or that the cells
may be responding to small amounts of endogenous GLP-1
secreted by the cells. In our transient assays in HIT cells there is no
evidence to suggest that the recombinant GLP-1R is
appreciably activated in the basal state.
The mechanisms whereby GLP-1 exerts its
insulinotropic activity have been under intense study in recent
years (reviewed in Ref. 1). Collectively, these studies suggest that
the actions of GLP-1 are multifaceted, with several
targets for action in the ß-cell. Targets and mechanisms include the
inhibition of ATP-sensitive K+ channels to facilitate cell
depolarization (12, 15), excitatory effects on VDCCs to increase
[Ca2+]i, including a suppression of
time-dependent inactivation (23, 24), and potentiation of activation of
L-type VDCC (23). Recent studies suggest that GLP-1 could
also exert an effect on insulin release at a level distal to an
elevation in [Ca2+]i (15, 25). Although there
may be several targets for GLP-1 action in the ß-cell,
pharmacological agents that inhibit the PKA pathway appear to negate
the effects of GLP-1 on KATP, VDCC, or events
distal to cell depolarization and Ca2+ influx.
Increases in cAMP accumulation and insulin secretion in response to
GLP-1 were not significant in control cells. These data
are in contrast to studies by Lu et al. (13), who
showed an increase in cAMP accumulation and insulin secretion in
HIT-T15 cells. Although the cells used in the previous study were not
examined for GLP-1R expression, it is highly likely that
the level of receptors would be considerably higher than we report in
the present study. Our data support previous studies, which found that
functional GLP-1Rs are indeed expressed in our HIT-T15
cell line, but the level of expression is extremely low (Fig. 1
). This
low level of GLP-1R expression made this insulin-secreting
cell line ideal for the present overexpression studies. Indeed,
GLP-1 (10 nM) elicited a large cAMP response
(Fig. 2C
) in cells expressing WT-GLP-1R that was
accompanied by a concomitant increase in insulin secretion (Fig. 2D
).
These data demonstrate a strong correlation between cAMP accumulation
induced by GLP-1 and insulin secretion. Increases were not
observed in cells expressing either of the mutant receptors.
GLP-1 treatment had no effect on inward currents in the
absence of glucose (control or WT-GLP-1R cells; see Fig. 6
).
Since previous reports demonstrated that GLP-1 had direct
stimulatory effects on voltage-clamped L-type VDCC in rat ß-cells
(23, 24), we conducted a series of experiments to compare the effects
of GLP-1 on voltage-clamped L-type VDCC activity in
WT-GLP-1R, mutant (DM-1) GLP-1R, and control
(GFP expressing only) cells. Similar to the findings of Suga et
al. (23), GLP-1 had signficant stimulatory effects on
L-type VDCC activity, acting to increase peak VDCC current amplitude
and shift the current-voltage relationship leftward in
WT-GLP-1R-expressing cells (Fig. 6
). In fact, we observed
a 142% increase in VDCC current magnitude with 10-8
M GLP-1 in WT-GLP-1R cells, an
effect that was much larger than the 30% increase with 2 x
10-8 M GLP-1 reported by Suga
et al. (23). This finding suggests that the increased
GLP-1R expression in WT-GLP-1R cells acts to
amplify the actions of GLP-1 on VDCC. In addition to
reported GLP-1-stimulated changes in time-dependent
inactivation, which act to slow VDCC inactivation (24),
GLP-1 caused a rightward shift in voltage-dependent
inactivation (Fig. 7
). This positive shift in inactivation would
contribute to increased VDCC availability and contribute to the
observed GLP-1-induced changes in the voltage dependence
of VDCC currents.
The lack of GLP-1 response in mutant and control cells was
not a result of nonfunctional VDCC expression, since the voltage
dependence or stimulatory effects of the L-type VDCC-specific agonist,
BAYK8644 (BAYK), were preserved and similar in all cells (Fig. 8
).
Since BAYK is not known to increase [cAMP]i (26), this
finding would support our hypothesis that AC uncoupling leads to the
abolished GLP-1 response in DM-1 cells. The slight upward
trend in the VDCC response to GLP-1 but its failure to
reach significance in control cells suggests that low
GLP-1R expression is likely responsible. This hypothesis
is supported by previous reports, which also demonstrated that the
actions of GLP-1 on VDCC are mediated through the AC
pathway via changes in [cAMP]i (11, 23, 24). Previous
studies in mouse ß-cells have demonstrated that elevation of
[cAMP]i or activation of PKA by forskolin (24, 27, 29)
caused little increase in VDCC current magnitude but caused a slowed
time course of inactivation. We find that GLP-1 caused
marked changes in VDCC current magnitude and voltage-dependent
inactivation in VDCC. In fact, the GLP-1-mediated changes
in VDCC activity we observed are similar to those reported for cardiac
myocyte VDCCs phosphorylated by PKA and protein kinase C (28, 29).
To further explore the mechanism of action of GLP-1 on
VDCC, we performed studies on the combinational effects of
GLP-1 and BAYK (Fig. 8
). BAYK is known to act at an
extracellular site on VDCC to cause VDCC current potentiation. Although
BAYK effects occur without elevation of [cAMP]i (26), its
potentiating effects are positively modulated by cAMP-dependent
phosphorylation (29). In fact, in a recent study on newborn rat cardiac
myocytes, the degree to which BAYK stimulated VDCC was found to be a
good indicator of the degree of VDCC phosphorylation in developing
myocytes (30). In our studies, the cumulative stimulatory effect of
GLP-1 and BAYK on VDCC activity was greater than either
drug added alone, irrespective of their order of addition. These
findings support the putative actions of GLP-1 to increase
[cAMP]i and positively modulate the actions of BAYK (Fig. 8
, A and B).
GLP-1R overexpression has been shown to increase glucose
responsivity and secretion in RIN cells in the absence of a
GLP-1 stimulus (21). Using reverse hemolytic plaque
assays, Rafizadeh et al. (21) demonstrated that this
increase in glucose-mediated secretion could be explained by an
increase in the number of glucose-responding cells. In contrast, islets
isolated from GLP-1R-deficient mice appeared to display
normal glucose responsivity (31). To examine glucose responsivity,
HIT-T15 cells expressing the WT-GLP-1R were examined and
compared with control cells and those expressing the cAMP-defective
mutant isoforms. Basal (0 mM glucose) insulin secretion
(Fig. 3
) was not found to differ among control, WT-GLP-1R,
IC31, and DM-1 transfection groups, and glucose either 5 or 10
mM elicited a characteristic 1.5- to 2-fold increase in
insulin secretion in all groups examined. This was supported by the
fact that mean resting membrane potentials were not significantly
different from that recorded for control, DM-1-, or WT-
GLP-1R-expressing cells. These results suggest that in the
absence of GLP-1, in HIT cells, the GLP-1R
remains primarily inactive and does not influence glucose competence.
They also suggest that in the case of RIN cells overexpressing the
GLP-1R (21), the possibility that the receptor prefers an
active conformation is supported, and that elevated cAMP accumulation
renders the cells more sensitive to glucose.
Binding of GLP-1 to its ß-cell receptor stimulates not
only insulin secretion but also increases insulin mRNA production (32),
likely via induction of insulin gene transcription through a
cAMP-dependent mechanism (33, 34). In keeping with this hypothesis,
mice deficient in the GLP-1R have reduced insulin gene
expression and reduced total pancreatic insulin content (35). In the
present study, insulin secretion rate over a 17-h culture period was
compared with total insulin content and insulin mRNA abundance, to
examine the effects of overexpression of the WT-GLP-1R and
the cAMP-defective mutants on insulin biosynthesis. Total insulin
content (Fig. 4C
) was similar in control and
WT-GLP-1R-transfected cells in the presence or absence of
a prolonged GLP-1 stimulus capable of significantly
elevating cAMP and insulin secretion. It is possible that the increased
secretion rate may have offset any increase in insulin synthesis
resulting from a GLP-1 stimulus. Northern blot analysis
and subsequent quantification (insulin/18 s, Fig. 4D
) revealed a small
but significant increase in proinsulin gene transcripts in cells
expressing WT-GLP-R in the presence of GLP-1 (10
nM), consistent with a role for GLP-1 on
insulin gene transcription. However, the correlation is not entirely
clear, since cAMP levels are maximal at 100 nM
GLP-1, when the increase in insulin mRNA did not reach
statistical significance. Perhaps future studies in models that will
allow a more prolonged expression of the DM-1 mutant isoform may allow
a more clear correlation between insulin gene transcription and
biosynthesis to be observed. Nevertheless, the present studies clearly
demonstrate that the GLP-1R efficiently couples to AC to
stimulate insulin secretion, actions mediated via effects on the VDCC.
Furthermore, receptors lacking critical residues in the proximal region
of the third intracellular loop can efficiently uncouple the receptor
from cAMP production, L-type VDCC action, and insulin secretion.
 |
MATERIALS AND METHODS
|
---|
Binding Assays
Synthetic human GLP-1(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) amide
(Bachem Torrance, CA) was used in all binding studies.
Radioiodination was accomplished by the chloramine-T method as
previously described (19). The
[125I]GLP-1(736 amide) product was
purified by reverse phase adsorption to a C-18 Sep-pak column (Waters
Associates, Milford, MA) and had a specific activity of approximately
125250 µCi/µg. Whole-cell binding assays were performed as
previously described (19). GLP-1R and GLP-1R
mutations were generated as previously described (18, 19). For binding
assays, 8 x 106 HIT-T15 cells (gift from Dr. Paul
Robertson, Pacific Northwest Research Institute, Seattle WA) between
passage 72 and 90 cultured in RPMI-1640 medium supplemented with 10%
FBS, 1% Penicillin/Streptomycin, and 1% L-glutamine were
seeded into 10-cm plates and transfected with 20 µg of expression
plasmids (pcDNA3 vector alone (control); WT-GLP-1R;
V331-I332-A333-deletion mutant (DM-1); and K334-L335-K336-deletion
mutant (IC31)] using Pfx1 lipid reagent (cells were incubated in the
transfection media for 3.54 h) according to the product
specifications (Invitrogen, Carlsbad CA). Binding assays
were carried out 48 h posttransfection. The cells were washed
twice in PBS and recovered from plates with 2 mM EDTA in
PBS. Cells (
1 x 106/tube) were incubated for 45
min at 37 C in binding buffer (RPMI containing 0.4% glucose, 1% BSA,
pH 7.4) with radiolabeled tracer [125I]GLP-1
amide (100,000 cpm,
270 pM) and unlabeled
GLP-1 at concentrations of 10-12 to
10-6 M, in a final volume of 200 µl. Cell
suspensions were centrifuged at 12,000 x g, and the
cell-associated radioactivity was counted (Cobra II, Canberra Packard,
Meriden, CT). Specific binding (total binding less nonspecific binding)
measured in the presence of excess (1 µM
GLP-1) was determined for the WT and each mutant receptor.
Binding characteristics, including specific binding and
IC50, were calculated from competitive binding-displacement
curves generated using curve-fitting software (Prism, GraphPad Software, Inc., San Diego, CA) as we have previously reported
(19).
Insulin and cAMP Assays
Twelve-well plates were seeded with 4 x 105
cells per well in a total volume of 1 ml. The cells were incubated for
48 h and then transfected with 2 µg of plasmid DNA (three wells
of pcDNA3 vector alone, one well for ß-gal plasmid, four wells for
WT-GLP-1R, four wells for DM-1 or IC31 mutant) using
Pfx1 as described above. After a 48 h posttransfection incubation
period, the culture media were replaced.
Insulin Assays
The following day (17 h), medium was replaced with 1 ml of Krebs-Ringer
buffer (KRB) containing 0.1% BSA (RIA grade), 0.238% (10
mM) HEPES, pH 7.4, and incubated twice for 30 min, after
which the cells were washed twice with 1 ml of KRB buffer and
then with 2 ml buffer. The final wash was replaced with 1 ml of
experimental buffer containing KRB, 0.1% BSA (RIA grade), 0.238% (10
mM) HEPES, pH 7.4, and 5 mM glucose and
stimulated with 10-8 M GLP-1 for
2 h. GLP-1 was prepared from lyophilized samples on
the day of assay and added from concentrated stocks. For
glucose-stimulation assays, the protocol remained the same with the
exception of the glucose concentration (0 mM, 5
mM, or 10 mM). For overnight secretion
experiments, the growth media were replaced after 48 h with media
containing 0, 10 nM, or 1 µM
GLP-1 and collected for assay after the 17-h test period.
In all cases, 700 µl of the experimental media from each well were
transferred to a microfuge tube and spun at 3000 rpm for 2 min. The top
300 µl of supernatant were transferred to a new tube and stored at
-70 C. Insulin RIAs were performed using rat insulin RIA kit from
Linco (St. Charles, MO). Total cell insulin content was determined
using acid extraction as previously described (36).
cAMP Assays
Immediately after media were collected for insulin RIA, the cells were
washed in cold PBS, and intracellular cAMP was extracted with 80%
ethanol. Lyophilized samples were reconstituted in sodium acetate
buffer (pH 6.2) and cAMP production was measured by RIA
(Biomedical Technologies, Stoughton, MA).
ß-Galactosidase assays were performed to assess transfection
efficiency on each multiwell plate using the manufacturers protocol
(Invitrogen, San Diego, CA). If efficiencies below 40%
were observed with ß-galactosidase, the cells were not used in
assays.
Northern Blot Analysis
Plates (10 cm) were seeded with 8 x 106
HIT-T15 cells and transfected with 20 µg of plasmid as described for
binding assays. After 48 h, the plates were washed twice with PBS,
and total cellular RNA was extracted using 2.5 ml of TRIzol reagent
(Life Technologies, Inc., Burlington, Ontario, Canada)
according to the protocol provided. Total RNA (25 µg) from HIT-T15
cells was suspended in sample buffer (6% formaldehyde, 50% formamide,
100 µl of 1x MOPS, 10% glycerol, and bromophenol blue). The RNA was
denatured and run on a 1.2% agarose-formaldehyde denaturing gel and
transferred to nylon membranes (Amersham Pharmacia Biotech, Oakville, Ontario, Canada) as previously described (37, 38). cDNA probes were prepared using the random primer labeling kit
from Life Technologies, Inc.. A partial hamster insulin
cDNA was obtained by RT-PCR on HIT-T15 cell RNA to yield a 350-bp
fragment corresponding to coding sequence. The probe used to detect
GLP-1R transcripts was a Kpn-I/HincII fragment
of the rat GLP-1R (kindly provided by Bernard Thorens,
Institute of Pharmacology and Toxicology, Lausanne, Switzerland) The
18S probe was generated as previously described (31). The blots were
hybridized at 40 C overnight as previously described (37, 38) and
washed with 0.5x SSC and 0.1% SDS at 55 C for 30 min. Densitometry
was performed as previously described to quantitate insulin transcripts
(35). Briefly, the autoradiogram was scanned and a constant size area
was used to convert the intensity of the bands to pixels.
Electrophysiological Assays
WT GLP-1R or DM-1 cDNAs lacking a stop codon were
generated by PCR where the primers were designed to amplify a product
where the stop codon was removed. This product was first cloned into
PCR 2.1 (Invitrogen) and then into the
HindIII-SmaI sites of pEBFP-N2 (CLONTECH, Palo
Alto, CA). Cells transfected with a WT-GLP-1 or the DM-1
mutant receptors tagged at the C terminus with EGFP. Control cells were
transfected with pEBFP-N2 alone. All cells were lightly trypsinized
(0.05% trypsin), washed with extracellular solution, and allowed to
equilibrate and adhere to a patch clamp study chamber that was mounted
on an inverted microscope (CK-2, Olympus Corp., Lake
Success, NY). Membrane currents through L-type VDCC were recorded using
standard whole-cell patch clamp techniques. To block K+
flux and observe large inward currents through L-type VDCCs, which were
not prone to run-down, extracellular solutions were used that contained
the following: 20 mM BaCl2, 90 mM
NaCl, 5 mM CsCl, 1 mM MgCl2, 10
mM glucose, and 10 mM HEPES. Intracellular
solutions in which K+ was replaced with cesium were used:
75 mM Cs2-aspartate, 1 mM
MgCl2, 20 mM tetraethylammonium chloride
(TEA)-Cl, 5 mM EGTA, 4 mM ATP-Mg, and 20
mM HEPES. Patch pipettes were prepared from 1.5-mm
thin-walled borosilicate glass using a two-state patch-pipette puller
(model pp83, Nari-shige, Tokyo, Japan). Pipette tips were
fire polished to resistances of 45 M
. The current flow between the
pipette and the bath solution was compensated to achieve a zero
baseline before seal formation. Standard tight-seal recording
techniques for seal formation were used, and access to the interior of
the cell was obtained by further suction to rupture the patch
membrane.
All electrophysiological experiments were performed at 2224 C
according to Hamill et al. (39), on representative cells
expressing EGFP. Currents were measured with an Axopatch-1D patch clamp
amplifier (Axon Instruments, Foster City, CA), filtered with a Bessel
filter (-3 decibels at 1 kHz) and recorded online by a computer
(IBM PC) using pCLAMP (v.6) software (Axon Instruments). Whole-cell
capacitance was routinely measured measured by the intermittent initial
testing by cancellation of the capacity transient and measured on
average 10.5 ± 0.6 pF. The average series resistance was 5.2
± 1.5 M
. Neither cell capacitance nor series resistance was
electronically compensated. VDCC currents were elicited by a protocol
in which cells were incrementally depolarized in +20 mV steps that were
held for 200 ms from -70 mV to +70 mV from a holding potential of -70
mV and peak steady state currents were measured at 250 ms. Inward,
Ca2+ currents were assessed between drug additions by a
single depolarizing step to +10 mV. ß-Cells studied in this manner
could be routinely patch clamped for up to 1 h with no significant
change in cell currents or membrane potential. Steady state
inactivation curves were obtained using a two-pulse protocol. From a
holding potential of -70 mV, a 15-sec depolarizing conditioning pulse
to different voltages was followed by a 250-msec test pulse to the
voltage at which the maximal VDCC currents were obtained (+10 mV).
Conditioning and test pulses were separated by a 20-msec return to the
holding potential (-70 mV). Steady state inactivation curves were
normalized by dividing the current amplitude (I) during the test pulse
by the maximal amplitude obtained in the absence of a conditioning
pulse (Imax). The data were fitted with the Boltzmann
function using pClamp6 software: I/Imax = {1 +
exp[Vm-V1/2)/k}-1 where
I/Imax is the relative current, Vm is the
membrane potential, V1/2 the half-maximum voltage of
inactivation, and k is the slope factor. V1/2 and k were
determined for each current obtained from individual cells.
Stock solutions or lyophilized drugs tested were stored at -22 C with
each aliquot being defrosted once and used over a 6-h study period. All
drugs were added to the chamber in microliter volumes, and routine
controls with the vehicles used for dissolution were done to exclude
nonspecific effects of the diluent. GLP-1 was lyophilized
and distilled water added. BAYK8644 (+/-, Calbiochem, La
Jolla, CA) was prepared in dimethylsulfoxide to
10-3 M stock and diluted further to 10
µM.
Statistics
All values are expressed as the mean ± SEM of
at least three independent observations unless stated otherwise.
Statistical analysis was performed using one way ANOVA followed by
Tukeys postanalysis (InStat, GraphPad Software, Inc.,
San Diego, CA).
 |
FOOTNOTES
|
---|
Address requests for reprints to: Michael B. Wheeler, Department of Physiology, University of Toronto, 1 Kings College Circle, Toronto, Ontario, Canada M5S 1A8.
This work was funded by grants to M.B.W. from the Medical Research
Council of Canada (MT-12898) and the Canadian Diabetes Association, and
to H.Y.G. and M.B.W. from the Eli Lilly & Co./Banting and
Best Diabetes Centre Research Program.
Received for publication February 16, 1999.
Revision received April 14, 1999.
Accepted for publication April 26, 1999.
 |
REFERENCES
|
---|
-
Fehmann HC, Göke G, Göke B 1995 Cell and
molecular biology of the incretin hormones glucagon-like peptide 1 and
glucose-dependent insulin releasing polypeptide. Endocr Rev 16:390410[Medline]
-
Nauck MA, Heimesaat MM, Ørskov C, Holst JJ, Ebert R,
Creutzfeldt W 1993 Preserved incretin activity of glucagon-like peptide
1 [736 amide] but not of synthetic human gastric inhibitory
polypeptide in patients with type-2 diabetes mellitus. J Clin
Invest 91:3017[Medline]
-
Gutniak M, Ørskov C, Holst JJ, Ahrén B, Efendic S 1992 Antidiabetogenic effect of glucagon-like peptide-1 (736)amide in
normal subjects and patients with diabetes mellitus. N Engl J
Med 326:131622[Abstract]
-
Ritzel R, Ørskov C, Holst JJ, Nauck MA 1995 Pharmacokinetic,
insulinotropic, and glucagonostatic properties of GLP-1(736 amide)
after subcutaneous injection in healthy volunteers. Dose-response
relationships. Diabetologia 38:720725[CrossRef][Medline]
-
Turton MD, OShea D, Gunn I, Beak SA, Edwards CMB, Meeran
KJ, Choi SJ, Taylor GM, Heath MM, Lambert PD, Wilding JP, Smith DM,
Ghatei MA, Herbert J, Bloom SR 1996 A role for glucagon-like peptide-1
in the central regulation of feeding. Nature 379:6972[CrossRef][Medline]
-
Moens K, Heimberg H, Flamez D, Huypens P, Quartier E, Ling Z,
Pipleers D, Gremlich S, Thorens B, Schuit F 1996 Expression and
functional activity of glucagon, glucagon-like peptide 1, and
glucose-dependent insulinotropic peptide receptors in rat pancreatic
islet cells. Diabetes 45:257261[Abstract]
-
Ørskov C, Poulsen SS 1991 Glucagon-like
peptide-I-(736)-amide receptors only in islets of Langerhans.
Diabetes 40:12921296[Abstract]
-
Thorens B 1992 Expression cloning of the pancreatic ß cell
receptor for the gluco-incretin hormone glucagon-like peptide 1. Proc
Natl Acad Sci USA 89:86418645[Abstract]
-
Dillon JS, Tanizawa Y, Wheeler MB, Leng XH, Ligon BB, Rabin
DU, Warren HY, Permutt MA, Boyd III AE 1993 Cloning and functional
expression of the human glucagon-like peptide (GLP-I) receptor.
Endocrinology 133:19071910[Abstract]
-
Serge G, Goldring S 1993 Receptors for secretin, calcitonin,
parathyroid hormone (PTH)/PTH-related peptide, vasoactive intestinal
peptide, glucagon-like peptide 1, growth hormone-releasing hormone, and
glucagon belong to a newly discovered G-protein linked receptor family.
Trends Endocrinol Metab 4:309314
-
Drucker DJ 1998 Glucagon-like peptides. Diabetes 47:159169[Abstract]
-
Holz GG, Kuhtreiber WM, Habener JF 1993 Pancreatic ß-cells
are rendered glucose-competent by the insulinotropic hormone
glucagon-like peptide-1(737). Nature 361:362365[CrossRef][Medline]
-
Lu M, Wheeler MB, Leng XH, Boyd III AE 1993 The role of the
free cytosolic calcium level in ß-cell signal transduction by gastric
inhibitory polypeptide and glucagon-like peptide I(737).
Endocrinology 132:94100[Abstract]
-
Widmann C, Burki E, Dolci W, Thorens B 1994 Signal
transduction by the cloned glucagon-like peptide-1 receptor: comparison
with signaling by the endogenous receptors and b-cell lines. Mol
Pharmacol 45:10291035[Abstract]
-
Gromada J, Bokvist K, Ding W, Holst JJ, Nielsen JH, Rorsman P 1998 GLP-1(736) amide stimulates exocytosis in human pancreatic
B-cells by both proximal and distal regulatory steps in stimulus
secretion coupling. Diabetes 47:5765[Abstract]
-
Gromada J, Rorsman P, Dissing S, Wulff BS 1995 Stimulation of
the cloned human GLP-1 receptor expressed in HEK cells induces
cAMP-dependent calcium-induced calcium release. FEBS Lett 373:182186[CrossRef][Medline]
-
Widmann C, Dolci W, Thorens B 1996 Desensitization and
phosphorylation of the glucagon-like peptide-1 (GLP-1) receptor by
GLP-1 and 4-phorbol 12-myristate 13-acetate. Mol Endocrinol 10:6275[Abstract]
-
Takhar S, Gyomorey S, Su RC, Mathi SK, Li X, Wheeler MB 1996 The third cytoplasmic domain of the tGLP-1 (736 amide) receptor is
required for coupling to the adenylyl cyclase system. Endocrinology 137:21752178[Abstract]
-
Mathi SK, Chan Y, Li S, Wheeler MB 1997 Scanning of the GLP-1
receptor localizes G protein activating determinants primarily to the
N-terminus of the third intracellular loop. Mol Endocrinol 11:424432[Abstract/Free Full Text]
-
Heller R, Kieffer T, Habener JF 1996 Point mutations in the
first and third intracellular loops of the glucagon-like peptide-1
receptor alter intracellular signaling. Biochem Biophys Res Commun 223:624632[CrossRef][Medline]
-
Rafizadeh CM, Wang Y, Janczewski AM, Henderson TE, Egan JM 1997 Overexpression of glucagon-like peptide-1 receptor in an
insulin-secreting cell line enhances glucose-responsiveness. Mol Cell
Endocrinol 130:109117[CrossRef][Medline]
-
Chicchi G, Graziano MP, Koch G, Hey P, Sullivan K, Vicario P,
Cascieri MA 1997 Alterations in receptor activation and divalent cation
activation of agonist binding by deletion of intracellular domains of
the glucagon receptor. J Biol Chem 272:77657769[Abstract/Free Full Text]
-
Suga S, Kanno T, Nakano K, Takeo T, Dobashi Y, Wakui M 1997 GLP-1(736) amide augments Ba2+ current through L-type
Ca2+ channel of the rat pancreatic B-cell in a
cAMP-dependent manner. Diabetes 46:17551760[Abstract]
-
Britsch S, Drews P, Lang F, Gregor M, Drews G 1995 GLP-1
modulates Ca2+ current but not K+ ATP current
in intact mouse pancreatic ß cells. Biochem Biophys Res Commun 207:3339[CrossRef][Medline]
-
Ding WG, Gromada J 1997 Protein kinase A-dependent stimulation
of exocytosis in mouse pancreatic ß cells by glucose-dependent
insulinotropic polypeptide. Diabetes 46:615621[Abstract]
-
Kokubun S, Reuter H 1984 Dihydropyridine derivatives prolong
the open state of Ca channels in cultured cardiac cells. Proc Natl Acad
Sci USA 81:48244827[Abstract]
-
Ammala C, Ashcroft FM, Rorsman P 1993 Calcium-independent
potentiation of insulin release by cyclic AMP in single ß-cells.
Nature 363:356358[CrossRef][Medline]
-
Tsien RW, Bean BP, Hess P, Lansman JB, Nilius B, Nowycky MC 1986 Mechanisms of calcium channel modulation by ß-adrenergic agents
and dihydropyridine calcium agonist. J Mol Cell Cardiol 18:691710[Medline]
-
Tiaho F, Lory RP, Nerbonne JM, Nargeot. 1990 Cyclic-AMP-dependent phosphorylation modulates the sterospecific
activation of cardiac Ca channels by BAYK8644. Pflugers Arch 417:5866[Medline]
-
Gomez J-P, Fares N, Potreau 1996 Effects of Bay K 8644 on
L-type calcium current from newborn rat cardiomyocytes in primary
culture. J Mol Cell Cardiol 28:22172229[CrossRef][Medline]
-
Scrocchi LA, Marshall BA, Cook SM, Brubaker PL, Drucker DJ 1998 Identification of GLP-1 actions essential for glucose homeostasis
in mice with a disruption of GLP-1 receptor signaling. Diabetes 47:632639[Abstract]
-
Drucker DJ, Philippe J, Mojsov S, Chick WL, Habener JF 1987 Glucagon-like peptide I stimulates insulin gene expression and
increases cyclic AMP levels in a rat islet cell line. Proc Natl Acad
Sci USA 84:34343438[Abstract]
-
Wang YH, Egan JM, Raygada M, Nadiv O, Roth J,
Montrose-Rafizadeh C 1995 GLP-1 affects gene transcription and
mRNA stability of components of the insulin-secretory system in
RIN 104638 cells. Endocrinology 136:49104917[Abstract]
-
Fehmann HC, Habener JF 1992 Insulinotropic hormone
glucagon-like peptide-I(737) stimulation of proinsulin gene
expression and proinsulin biosynthesis in insulinoma ßTC-1 cells.
Endocrinology 130:15966[Abstract]
-
Pederson RA, Satkunarajah M, McIntosh CHS, Scrocchi L, Flamez
D, Schuit F, Drucker DJ, Wheeler MB 1998 Enhanced GIP secretion and
insulinotropic action in GLP-1 receptor -/- mice. Diabetes 17:10461052
-
Wheeler MB, Bouquillon T, Ghai M, Sheu L, Bennett MK, Trimble
W, Gaisano HY 1996 Characterization of SNARE protein expression in the
pancreatic ß-cell line ßTC6F7 and pancreatic islets.
Endocrinology 137:13401348[Abstract]
-
Wheeler MB, Nishitani J, Buchan AMJ, Kopin AS, Chey WY, Chang
TM, Leiter AB 1992 Identification of a transcriptional enhancer
important for enteroendocrine and pancreatic islet-specific expression
of the secretin gene. Mol Cell Biol 12:35313539[Abstract]
-
Wheeler MB, Lu M, Dillon J, Leng XH, Chen C, Boyd III AE 1993 Functional expression of the glucagon-like peptide-1(737) receptor:
evidence for coupling to phospholipase C as well as adenylyl cyclase.
Endocrinology 133:5762[Abstract]
-
Hamill OP, Neher B, Sigworth FJ 1981 Improved patch-clamp
techniques for high-resolution current recording for cells and
cell-free membrane patches. Pflugers Arch 391:85100[Medline]