From INSERM U376, CHU Arnaud-de-Villeneuve, 34295 Montpellier,
France and the University Laboratory of Physiology,
Oxford University, Parks Road, Oxford OX1 3PT, United Kingdom
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ABSTRACT |
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Using the MIN6 B-cell line, we investigated the
hypothesis that miniglucagon, the C-terminal (19-29) fragment
processed from glucagon and present in pancreatic A cells, modulates
insulin release, and we analyzed its cellular mode of action. We show that, at concentrations ranging from 0.01 to 1000 pM,
miniglucagon dose-dependently (ID50 = 1 pM) inhibited by 80-100% the insulin release triggered by
glucose, glucagon, glucagon-like peptide-1-(7-36) amide (tGLP-1), or
glibenclamide, but not that induced by carbachol. Miniglucagon had no
significant effects on cellular cAMP levels. The increase in
45Ca2+ uptake induced by depolarizing agents
(glucose or extracellular K+), by glucagon, or by the
Ca2+channel agonist Bay K-8644 was blocked by miniglucagon
at the doses active on insulin release. Electrophysiological
experiments indicated that miniglucagon induces membrane
hyperpolarization, probably by opening potassium channels, which
terminated glucose-induced electrical activity. Pretreatment with
pertussis toxin abolished the effects of miniglucagon on insulin
release. It is concluded that miniglucagon is a highly potent and
efficient inhibitor of insulin release by closing, via
hyperpolarization, voltage-dependent Ca2+
channels linked to a pathway involving a pertussis toxin-sensitive G protein.
Like many other polypeptide hormones (1-3), glucagon is processed
from a large precursor, the 160-amino acid proglucagon produced in the
A-cells of the islets of Langerhans, in the L cells of the intestinal
mucosa, and in specialized neurons of the central nervous system
present mainly in the hypothalamus and in the medulla oblongata (4).
Glucagon is known for its hyperglycemic activity through its action on
liver via a seven-transmembrane domain receptor linked to adenylyl
cyclase via a GTP-binding protein of the Gs sub-type (5). At the level
of its target tissues such as the liver, glucagon is partially
processed through a cleavage at the Arg17-Arg18
basic doublet by a cell surface protease referred to as
"miniglucagon-generating endopeptidase"
(MGE)1 (6) leading to the
production of a C-terminal (19-29) fragment called "miniglucagon"
(7-9). Miniglucagon, which does not interfere with the adenylyl
cyclase activity, inhibits at picomolar concentrations the hepatic
plasma membrane calcium pump (10). On cultured cardiac myocytes,
miniglucagon was shown to potentiate at nanomolar concentrations the
positive inotropic effect of glucagon, whereas, when used alone at
picomolar concentrations, it displayed a negative inotropic effect on
myocyte contraction (11). These observations suggested a new role for
glucagon as a prohormone and a biological role for miniglucagon as a
daughter hormone that modulates the effects of the mother hormone (12).
On the other hand, pancreas is the only known tissue in which
miniglucagon is present in a stored form, at molar concentrations in
the range of 2-5% of that of glucagon (13).
In view of preliminary results suggesting that miniglucagon is able to
inhibit glucose- and glucagon-induced insulin release (12), we studied
the ability of miniglucagon to modulate secretagogue-induced insulin
release using the MIN6 cell line which displays characteristics that
compare well with that of normal We show here that miniglucagon, in a concentration range (starting at
10 Peptides and Chemicals
Nle27 miniglucagon was synthesized in our laboratory
(15). Synthetic glucagon-like peptide-1-(7-36) amide (GLP-1-(7-36)
amide) was obtained from Peninsula Laboratories (San Carlos, CA),
glucagon from Novo Research Institute (Bagsvaerd, Denmark),
glibenclamide from Guidotti Spa (Pisa) Laboratory, and somatostatin
from Neosystem. Radioimmunoassay of insulin was performed using
125I-porcine insulin, rat insulin (Novo, Denmark) as
standard, and the guinea pig anti-porcine insulin antibody 41 previously described (16). 45Ca was obtained from NEN Life
Science Products (France). Bay K-8644 was purchased from
Calbiochem-Novabiochem (La Jolla, CA), and nifedipine was from Sigma.
Methods
Cell Culture--
MIN6 cells were originally obtained from Dr.
H. Ishihara (Tokyo, Japan). The cells were grown in Dulbecco's
modified Eagle's medium containing 25 mmol/liter glucose (DMEM, Life
Technologies, Inc.) supplemented with 15% fetal calf serum (Life
Technologies, Inc.), 100 µg/ml streptomycin (Life Technologies,
Inc.), 100 units/ml penicillin sulfate (Life Technologies, Inc.), and
75 nM Insulin Release--
MIN6 cells were plated in 1 ml of DMEM (25 mM glucose) in 24-well plates at a density of 1 × 106 cells per well for 3-5 days. Insulin release was
determined using a static incubation method in 5% CO2,
95% air at 37 °C with cells in exponential growth. The medium
culture was changed 18 h before the experiments. Insulin secretion
from MIN6 cells monolayers was performed in HEPES-balanced Krebs-Ringer
bicarbonate buffer (119 mmol/liter NaCl; 4 mmol/liter KCl; 1.2 mmol/liter KH2PO4; 1.2 mmol/liter
MgSO4; 2.5 mmol/liter CaCl2; 20 mmol/liter
HEPES, pH 7.5) containing 0.5% BSA (KRB buffer).
The day of the experiment, the medium was removed and the cells were
washed twice with 500 µl of KRB buffer. Cells were preincubated for
1 h in 500 µl of KRB buffer containing 1 mmol/liter glucose in
5% CO2, 95% air at 37 °C. This buffer was removed, and
MIN6 cells were then preincubated for 2 h in 500 µl of KRB
buffer containing varying concentrations of glucose and other test
agents. At the end of the incubation period, media were collected, and
floating cells, if any, were eliminated by centrifugation at 1000 rpm
for 5 min.
Radioimmunoassay of Insulin--
Insulin in supernatants was
measured by radioimmunoassay as described previously (16), using
125I-insulin, rat insulin standard, and anti-insulin
porcine antiserum. Briefly, the assay was performed in a final volume
of 500 µl of 0.025 M borate buffer containing 0.5% BSA.
After a 4-day incubation at 4 °C, separation of free insulin was
realized using activated charcoal (5 g/100 ml in 0.1 M
borate buffer) after addition of 100 µl/sample horse serum. After
dilution and centrifugation at 3000 rpm for 5 min, the pellet was
counted in a Determination of cAMP Production--
MIN6 cells were grown in
24-well plates for 3-5 days under the same conditions as for insulin
release. The medium was changed 1 day before the experiments. On the
day of the experiment, cells were washed twice with DMEM containing 4.5 mmol/liter glucose without fetal calf serum before the addition of 500 µl of DMEM buffer supplemented with 1% BSA and 1 mM IBMX
as an inhibitor of cyclic AMP phosphodiesterase and containing the test
substances. After a 15-min incubation at room temperature, the cells
were extracted using 60% perchloric acid, the sample was neutralized with 9 N KOH succinylated to increase the sensitivity of the assay (17), and cyclic AMP was quantified by radioimmunoassay.
Measurement of 45Ca2+ Influx--
24 h
before the experiment, the culture medium was changed. On the day of
the experiment, the cells were washed twice with 500 µl of KRB buffer
and preincubated for 30 min at 37 °C in 250 µl of KRB buffer
containing 1 mmol/liter glucose in 5% CO2, 95% air. The
preincubation solution was then replaced by 250 µl of KRB containing
8 µCi/ml 45CaCl2 (Amersham Pharmacia Biotech,
UK; 5-50 mCi/mg Ca) and the test agents. The reaction, developed at
37 °C, was stopped by aspiration of the medium. The cells were
rapidly washed four times with ice-cold buffer (135 mM
NaCl, 5 mM KCl, 2.5 mM CaCl2, 1 mM lanthanium chloride, 10 mM HEPES). The cells
were then solubilized in 1 ml of KRB containing 0.1% Triton X-100 for
1 h at room temperature. An aliquot of the solution (100 µl) was
then assayed for 45Ca2+ content in a
Electrophysiology--
Cells were plated onto plastic Petri
dishes and maintained in tissue culture prior to use. For whole-cell
voltage clamp, cells were chosen that were rounded and apparently
single. For current-clamp recording, both single cells and clusters of
cells were used. To maintain cell metabolism and second-messenger
systems intact, membrane currents and potential were recorded using the
perforated-patch whole-cell technique as described previously (18, 19).
Ca2+ currents were measured using voltage clamp with a bath
solution that contained (in mM) 108 NaCl, 30 TEACl, 5.6 KCl, 1.2 MgCl2, 2.6 CaCl2, 10 HEPES (pH 7.4 with NaOH), and 10 glucose
and 0.1% w/v bovine serum albumin. The pipette solution contained (in
mM) 76 Cs2SO4, 10 KCl, 10 NaCl, 1 MgCl2, and 10 HEPES (pH 7.2 with CsOH). Perforation was obtained by the addition of
0.2 µg ml-1 amphotericin B to the pipette solution and
was considered adequate when the series conductance was > 40 nS
(mean 69 ± 8 nS, n = 10). Inward Ca2+
currents were elicited by stepping the membrane to potentials positive
to Statistical Analysis--
Results were analyzed by Student's
t test for unpaired data.
Effects of Glucose, Glucagon, and tGLP-1 on Insulin
Release--
We first analyzed the response of our batches of MIN6
cells to classical secretagogues, namely glucose (an indirect
depolarizing agent), glucagon, and tGLP-1 (potentiators of
glucose-induced insulin release via a cyclic
AMP-dependent pathway), and the sulfonylurea glibenclamide
(a blocker of ATP-dependent potassium channels). These
molecules increased, at concentrations similar to that observed in
other biological models, insulin release from this beta cell line:
glucose, at 20 mM, induced a 6-fold increase in this
parameter as compared with base line measured at 1 mM
glucose, the half-maximal response being achieved at around 10 mM (Fig. 1,
inset). As shown in Fig. 1, insulin release was also
dose-dependently stimulated by glucagon (EC50 = 10.1 ± 0.15 nM) and tGLP-1 (EC50 = 1.32 ± 0.2 nM), as well as (data not shown) by the
sulfonylurea glibenclamide (EC50 Effects of Miniglucagon on Glucose, Sulfonylurea-stimulated Insulin
Release--
As shown in Fig.
2A, miniglucagon
dose-dependently inhibited the 10 mM
glucose-induced insulin release. The threshold dose for the
miniglucagon effect was observed between 0.01 and 0.1 pM,
EC50 was around 2 pM, and a virtually complete
inhibition was noted at 1 nM miniglucagon. In Fig.
2B, it may be seen that miniglucagon also
dose-dependently inhibited 20 nM
glibenclamide-induced insulin release in a range starting in the
sub-picomolar concentrations with an IC50 around 0.3 pM.
Effects of Miniglucagon on Glucagon- and tGLP-1-stimulated Insulin
Release--
In Fig. 3, A and
B, it may be seen that miniglucagon also
dose-dependently inhibited 10 nM
glucagon-induced or 10 nM tGLP-1-induced insulin release,
again at threshold doses between 0.01 and 0.1 pM.
Half-maximal effects were noted at concentrations close to 1 pM, and again, a very large proportion (around 90%) of the
stimulated secretion was suppressed at 1 nM miniglucagon
(Fig. 3, A and B).
Because glucagon and tGLP-1 are known to stimulate insulin release
via a cyclic AMP-dependent pathway (20), we
determined whether miniglucagon might modify the B-cell cyclic AMP content.
Effect of Miniglucagon on Glucagon- and tGLP-1-stimulated Cyclic
AMP Production--
Both tGLP-1 and glucagon stimulated by a 2-fold
factor cyclic AMP production in MIN6 cells in a
concentration-dependent manner (Fig.
4). As expected from the data obtained
when insulin release was studied, glucagon (EC50 = 3.17 ± 0.05, n = 4) was less potent than tGLP-1
(EC50 = 1.34 ± 0.1, n = 4) (Fig. 4).
In contrast, miniglucagon did not display any effect on basal or on
glucagon- or tGLP-1-stimulated cyclic AMP production. These results
clearly indicate that miniglucagon inhibits both glucagon- and tGLP-1- stimulated insulin release without interfering with the cyclic AMP
pathway.
Effect of Miniglucagon on Cholinergic Potentiation of Insulin
Release--
Because glucose, sulfonylureas, glucagon, and tGLP-1 have
the common ability to induce calcium entry, we determined whether miniglucagon might also inhibit insulin release stimulated by a
secretagogue known to act mainly via a calcium release from intracellular stores. For that purpose, we used the cholinergic agonist
carbachol, known to potentiate glucose-induced insulin release. As
shown in Fig. 5, miniglucagon was unable
to modify the 10 µM (Fig. 5A) or the 1 µM carbachol-induced (Fig. 5B) insulin release
in the range of 0.01 to 100 pM, which was shown to deeply affect the stimulatory effect of glucose, sulfonylurea, glucagon, or
tGLP-1. However, a significant inhibition was noted of the highest (1 nM) miniglucagon dose both at 1 and 10 µM
carbachol.
It is apparent from all these data that miniglucagon acts on the beta
cell through a pathway that is shared by glucose, glucagon, tGLP-1, and
sulfonylureas but not by carbachol. Because calcium influx through L
type voltage-dependent Ca2+ channels (VDCC) has
a key role in secretagogue-induced insulin release (29), VDCC are a
good candidate for being this common step. We therefore tested the
modulating effect of miniglucagon on Ca2+ influx triggered
by the above secretagogues.
Effect of Miniglucagon on Stimulated Ca2+
Influx--
As shown in Fig.
6A, miniglucagon
dose-dependently inhibited 10 mM
glucose-induced Ca2+ influx with an IC50 around
1 pM. Similarly, miniglucagon totally suppressed the
potentiating effect of 10 nM glucagon plus 10 mM glucose-induced Ca2+ influx (Fig.
6B). Miniglucagon displayed significant effects at doses as
low as 0.1 pM on these Ca2+ influx. Taken
together, our findings strongly suggested that miniglucagon inhibits
insulin release by blocking the secretagogue-induced Ca2+
influx into the beta cells.
Given the importance of VDCC in secretagogue-induced insulin release
(23, 29), we hypothesized that miniglucagon might affect the behavior
of this particular channel. To address this issue, we designed the
following series of experiments. First, an opener of L type
Ca2+ channels, Bay K-8644 (24), was used to open the
channel under conditions of 10 mM glucose stimulation. As
shown in Fig. 7A, miniglucagon
was able in the range 0.01 pM to 1 nM to
totally suppress the Bay K-8644-induced Ca2+ influx,
displaying significant effects at doses as low as 0.01 pM.
For comparison, 1 nM miniglucagon was as effective as 2 µM nifedipine (Fig. 7A), a direct inhibitor of
L type Ca2+ channels (25). As additional proof of the
identity of the type of Ca2+ channel involved in the
miniglucagon action and because opening of voltage-sensitive
Ca2+ channels may be obtained by membrane depolarization,
we analyzed whether miniglucagon was able to suppress a
potassium-induced Ca2+ influx similar in size to that
induced by physiological insulin secretagogues. As shown in Fig.
7B, miniglucagon, in the 0.01 pM to 1 nM dose range, almost totally suppressed the 10 mM potassium-induced Ca2+ influx.
The above data suggest that the inhibitory effect of miniglucagon
on insulin release is because of closure of L type
voltage-dependent Ca2+ channels. To address the
hypothesis of a direct or an indirect effect of the miniglucagon on
these calcium channels, we tested the effect of miniglucagon 1) on L
type Ca2+ currents and 2) on the MIN6 cell electrical
activity that controls Ca2+ influx (26-29), using the
whole-cell patch-clamp method.
Electrophysiology, Ca2+ Currents--
Inward
Ca2+currents were elicited at potentials positive to
After 5 min of perfusion of 10 Electrophysiology, Membrane Potential--
We next investigated
the effect of the peptide on the membrane potential and electrical
activity induced by glucose. The electrical activity of the beta cell
results from the complex interplay of several different ionic
conductances (29). Therefore, changes in electrical activity can be
used as a sensitive detector of the effect of drugs and hormones on ion
channels. Furthermore, glucose-induced insulin secretion is directly
controlled by the Ca2+ influx that results from the
Ca2+-dependent action potential activity
induced by this secretagogue. Therefore, any minor change in ion
channel behavior and electrical activity will have major effects on
insulin secretion.
In the absence of exogenous metabolite, the membrane potential of the
MIN6 cell was electrically silent with a mean value of
In three cells tested, 10 Effect of Pertussis Toxin Pretreatment of MIN6 Cells on
Miniglucagon Action--
It is known from other studies (32) that
miniglucagon is active through a specific receptor linked to at least
one type of GTP binding protein (G protein). To determine the type of G protein involved in the action of miniglucagon on beta cells, we
analyzed whether pretreating MIN6 cells with pertussis toxin would
modify the effectiveness of miniglucagon on secretagogue-induced insulin release. First, both control and toxin-pretreated MIN6 cells
(200 ng/ml, overnight) responded to an elevation of glucose from 1 to
25 mM with an increased rate of insulin release, although toxin-pretreated cells consistently showed a higher secretory rate at
glucose 10 mM (data not shown). An overnight pretreatment of the cells with 200 ng/ml pertussis toxin completely suppressed the
inhibitory effect of 0.1 µM somatostatin and 1 nM miniglucagon on 10 mM glucose-induced
insulin release (Fig. 10). Because it was shown that adrenaline may activate, via the Within the general mechanisms of proglucagon processing leading to
various peptide fragments with different biological roles (7),
processing of glucagon into miniglucagon at its
Arg17-Arg18 doublet displays many
singularities. (i) The site is not a typical processing site for
prohormone convertases (PCs) which use mostly Lys-Arg or Arg-Lys
doublets (33, 34) or more complex combinations such as the
R-X-K/R-R used by furin (35). On the other hand, we showed
that glucagon is cleaved at its dibasic site by an original protease
referred to as MGE, which was isolated from liver membranes (6) and
characterized as a 100-kDa protein (6). The C-terminal product of the
reaction, glucagon 19-29 or "miniglucagon," displays original
features, in particular it modulates the hepatic plasma membrane
calcium pump (10) and plays a role in the inotropic and chronotropic
action of glucagon (11). Its activity is mediated by a specific
receptor that remains to be characterized but that is already known to
be linked to G proteins (32). (ii) The miniglucagon action is observed
at picomolar concentrations, that is 2 to 3 orders of magnitude lower
than the active concentrations of glucagon, the mother hormone. (iii)
The strikingly high clearance rate of the peptide from circulation,
mostly because of its very rapid degradation by the liver (8, 13),
precludes any hormonal status. Accordingly, a role as a "daughter
peptide", released locally from circulating glucagon and modulating
the action of the mother hormone just before being degraded, was
established (7, 12). On the other hand, the presence of miniglucagon in
pancreas (13) and in a pancreatic alpha cell
line2 at molar concentrations
ranging from 2 to 5% of that of glucagon suggested a role for
miniglucagon in islet physiology. Our study was designed in light of
this background.
As an in vitro model of pancreatic beta cell, we used the
MIN6 cell line which displays most of the features of authentic beta
cells, in particular the graded release of insulin in response to
physiological concentrations of glucose (14) and to several other
insulin-secretagogues. Here we show that miniglucagon is a highly
potent and efficient inhibitor of insulin release. In view of the
miniglucagon concentrations in the alpha cells (2 orders of magnitude
lower than that of glucagon) and if we suppose, as a working
hypothesis, that glucagon and miniglucagon are released at the same
rate, then it is logical to expect an effect of miniglucagon in the
10 Miniglucagon has the characteristics of a very potent and efficient
inhibitor of insulin release triggered by secretagogues known to act
largely via calcium influx through voltage-dependent calcium channels. These channels may be activated by membrane depolarization induced either by changes in extracellular
K+ concentration or via closure of
KATP channels (e.g. glucose and sulfonylurea), by cyclic AMP-dependent protein kinase
phosphorylation (e.g. glucagon and tGLP-1), or by direct
action of a pharmacological agent (e.g. Bay K-8644). We
report here that miniglucagon is able to completely inhibit calcium
uptake elicited by glucose, high extracellular K+,
glucagon, or tGLP-1 and Bay K-8644. It was thus of particular importance to determine how miniglucagon acts on calcium channels. First, as miniglucagon is unable to modify cyclic AMP levels, we can
conclude that the peptide does not modulate L-type
voltage-sensitive Ca2+ channels via cyclic
AMP-dependent protein kinase phosphorylation. Another
possibility was that miniglucagon acts directly on Ca2+
channels via a G protein, as shown for acetylcholine (37). However,
from electrophysiological experiments, we were able to determine that
the peptide does not have a direct effect on the beta cell
Ca2+ channels. Third, miniglucagon was able to alter
transmembranous cationic fluxes, as manifested by hyperpolarization,
and to decrease the incidence of spike activity elicited by glucose.
This provides a strong indication that miniglucagon acts by opening
potassium channels, triggering a membrane hyperpolarization and thus
suppressing the ability of secretagogues to open voltage-sensitive
Ca2+ channels. It is noteworthy that, similarly to
somatostatin, galanin or adrenaline (30, 38), the miniglucagon receptor
is linked to its main effector (probably the potassium channels) via a
pertussis-sensitive G protein. On the other hand, one of the
originalities of miniglucagon is that it has no effect on cyclic AMP,
indicating that a peptide may trigger a deep and long term inhibition
of insulin release without modifying this parameter and suggesting that
the miniglucagon receptor interacts with a G protein, or a set of G
proteins that differ from that interacting with somatostatin and
galanin receptors.
Determining the precise type of G protein involved in the miniglucagon
action, characterizing the beta cell miniglucagon receptor linked to
this G protein and analyzing the physiological conditions of
miniglucagon release inside the islets, in a dependent or independent manner as compared with that of glucagon, are new issues in the development of this research on this newly discovered potential physiological mechanism by which an islet cell may control insulin release. Analysis of this mechanism in pathological states may also
shed a new light on particular aspects of diabetes mellitus.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cells (14), in particular a
response to glucose in the physiological range. We have also explored
the intracellular pathway through which miniglucagon inhibits insulin release.
14-10
13 M) which fits with
the amount of peptide presumably present in the extracellular medium
within the islets, is able to suppress by 80-100% the insulin release
stimulated by molecules known to open voltage-dependent
calcium channels such as glucose, glucagon, tGLP-1, or glibenclamide,
but not the insulin release stimulated by carbachol, a molecule known
to increase InsP3 and cytosolic Ca2+.
Miniglucagon had no effect on the cellular cyclic AMP levels but
suppressed secretagogue-induced calcium entry. Miniglucagon induced a
hyperpolarization of the membrane potential and thus probably inhibited
the action of all those secretagogues via an indirect
inhibition of the voltage-dependent L-type
calcium channels. The miniglucagon action on insulin release was
suppressed after pre-treatment of the cells with pertussis toxin. It is
proposed that miniglucagon, present in pancreatic A-cells, acts as a
local inhibitory regulator of insulin release by turning off the main external calcium source for
cells via a specific receptor linked, through a pertussis toxin-sensitive GTP-binding protein, to ion channels that control the cell polarity.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol (Sigma), equilibrated with 5%
CO2, 95% air at 37 °C. DMEM was changed every 48 h
of culture. When reaching 80% confluence, the cells were detached by
treatment with 2.5% trypsin, 0.5 mM EDTA. MIN6 cells used
in the present study were harvested at passages 15 to 25.
-scintillation counter (LKB-Wallac).
-counter after addition of a liquid scintillation medium (Complete
Phase Combining System, Amersham Pharmacia Biotech).
60 mV for 250 ms at a frequency of 0.1 Hz. The holding potential
was
70 mV. Currents flowing because of leak conductances and
uncompensated capacitance were removed by subtracting the scaled
average of currents elicited by voltage steps to
60,
80, and
90
mV. To control for variation in cell size, currents have been
normalized to the cell capacitance (mean 7.3 ± 0.5 picofarad, n = 10). Membrane potential was monitored using current
clamp with the same solutions used for the measurement of
Ca2+ currents except that TEACl in the bath was replaced
with NaCl, glucose was added as required, and Cs+ in the
pipette was replaced with K+. Currents and potentials are
referenced to the pipette in the bath. No corrections have been made
for liquid junction potentials (<4 mV) or series resistance errors
(<2 mV). All experiments were conducted at 32 ± 1 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 nM),
all experiments being run in the presence of 10 mM
glucose.
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Fig. 1.
Glucose-, glucagon-, and tGLP-1-stimulated
insulin release from MIN6 cells. Insulin release from MIN6 cells
in culture was measured after incubation with the corresponding
secretagogue as described under "Experimental Procedures." Data are
means ± S.E. for fifteen determinations (inset) or
twelve determinations.
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Fig. 2.
Effects of miniglucagon on glucose- and
sulfonylurea (glibenclamide)-stimulated insulin release.
A, dose-response curve of miniglucagon on 10 mM
glucose-stimulated insulin release. Data are means ± S.E. of
three to four experiments, each performed in triplicate. B,
dose-response curve of miniglucagon on 10 mM glucose and 20 nM glibenclamide. Data are means ± S.E. of two
experiments, each performed in triplicate. Statistical significance was
determined by comparing the data obtained in the presence and in the
absence of miniglucagon. *, p < 0.05; **,
p < 0.01; ***, p < 0.001.
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Fig. 3.
Effect of miniglucagon, glucose+, glucagon ,
or glucose + tGLP-1-stimulated insulin release. Shown is the
dose-response curve of miniglucagon on 10 mM glucose + 10 nM glucagon-stimulated insulin release (A) and
on 10 mM glucose + 10 nM tGLP-1-stimulated
insulin release (B). Data are means ± S.E. of three to
four experiments, each performed in triplicate. Statistical
significance was determined by comparing the data obtained in the
presence and in the absence of miniglucagon. **, p < 0.01; ***, p < 0.001.
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Fig. 4.
Effects of miniglucagon on basal, glucagon-,
or tGLP-1- stimulated cyclic AMP production in MIN6 cells. Data
are means ± S.E. of three experiments, each performed in
triplicate.
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Fig. 5.
Effect of miniglucagon on 10 and 1 µM carbachol-stimulated insulin
release. Dose-response curve of miniglucagon on 10 µM carbachol-stimulated insulin release (A)
and on 1µM carbachol-stimulated insulin release
(B), in the presence of 3 mM glucose. Data are
means ± S.E. of two experiments, each performed in triplicate.
Statistical significance was determined by comparing the data obtained
in the presence and in the absence of miniglucagon. *,
p < 0.05; **, p < 0.01.
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Fig. 6.
Effects of miniglucagon on glucose- and
glucagon-stimulated calcium uptake from MIN6 cells.
45Ca2+ uptake was measured as described under
"Experimental Procedures." The test agents were present
simultaneously. A, 10-min incubation with 10 mM
glucose and with or without various concentrations of miniglucagon;
B, 10-min incubation with 10 nM glucagon in the
presence of 10 mM glucose. Data are means ± S.E. of
nine determinations (A) and six determinations
(B). Statistical significance was determined by comparing
the data obtained in the presence and in the absence of miniglucagon.
**, p < 0.01; ***, p < 0.001.
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Fig. 7.
Effects of miniglucagon on
45Ca2+ uptake triggered
by opening of voltage-dependent Ca2+
channels. 45Ca2+ uptake was measured
as described under "Experimental Procedures." The test agents were
present simultaneously. A, 3-min incubation with 10 mM glucose and a fixed (2 µM) concentration
of Bay K-8644 with or without various concentrations of miniglucagon or
2 µM nifedipine. Data are means ± S.E. of nine
determinations. B, 3-min incubation with 3 mM
glucose and 10 mM KCl with or without various
concentrations of miniglucagon. Data are means ± S.E. of six
determinations. Statistical significance was determined by comparing
the data obtained in the presence and in the absence of miniglucagon
(panels A and B). *, p < 0.05;
**, p < 0.01; ***, p < 0.001.
60
mV. They were characterized by a rapid rising phase that peaked and
then slowly inactivated to a steady state level (Fig.
8A). Both the peak- and steady
state current-voltage relationships are bell-shaped. The maximum inward
current occurs at approximately +10 mV and reverses at potentials
positive to +40 mV (Fig. 8C). The relationship between
integrated Ca2+ entry and voltage mirrors that of current
with voltage. Maximum Ca2+ influx occurred at approximately
+10 mV (Fig. 8D).
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Fig. 8.
Calcium currents. Panels A
and B, representative perforated patch whole-cell recordings
of Ca2+ currents from single MIN6 cells. Currents were
elicited by 250-ms pulses from a holding potential of 70 mV to
potentials of:
40,
30,
20,
10, and 0 mV (i); +10, +20, +30, and
+40 mV (ii). A, Ca2+ currents recorded in
control. B, Ca2+ currents recorded after 5 min
in 10
10 M miniglucagon. C, mean
peak (squares) and steady-state (circles)
current-voltage relationships for Ca2+ currents recorded in
control (filled symbols) and after 10
10
M miniglucagon (open symbols) (n = 7). D, mean integral Ca2+ entry recorded in
control (filled symbols) and after 10
10
M miniglucagon (open symbols) (n = 7).
10 M
miniglucagon, a concentration that inhibits insulin secretion by 93%
(Fig. 2A), the Ca2+ currents remained unchanged
in seven cells tested (two examples displayed in Fig. 8B).
Neither the peak nor the steady state current-voltage relationship were
affected by the hormone (Fig. 8C). The integral Ca2+ entry was also unaffected by the peptide (Fig.
8D). A lack of effect of 10
10 M
miniglucagon on Ca2+ currents was also observed in single
beta cells isolated from normal mice (data not shown, n = 4). These data indicate that miniglucagon does not reduce
Ca2+ influx and insulin secretion from MIN6 cells by a
direct block of the Ca2+ channels. Somatostatin, at a
concentration (10
9 M) that inhibits insulin
secretion by 45% was also without effect on Ca2+ currents
(data not shown, n = 2), as previoulsy shown in beta cells isolated from normal mice (30).
65 ± 1 mV (n = 18). In 19 cells tested, the membrane potential began to depolarize within 33 ± 13 s (n = 11) of the addition of 10 mM glucose. After 51 ± 4 s, the membrane potential was sufficiently depolarized
(
48 ± 2 mV, n = 8) to evoke electrical activity
(Fig. 9A). In the majority of
cases, this consisted of continuous firing of action potentials.
However in three instances, the clustering of action potentials into
burst-like electrical activity was observed. These were reminiscent
of the typical bursting behavior observed in the intact islet
(data not shown) (29, 31). In three of nine cells tested,
10
10 M miniglucagon reduced the frequency
of action potential firing. In four cells tested, 10
9
M miniglucagon consistently caused the membrane potential
to hyperpolarize, which terminated the electrical activity. The
membrane potential hyperpolarized to
64 ± 4 mV, a value close
to that found in the absence of glucose (
68 ± 2 mV for the same
cells). The hyperpolarization was also associated with a decrease in
voltage noise and a reduction in the input resistance of the cell.
Taken together these data are consistent with miniglucagon activating a
potassium conductance. A 10-fold high dose of miniglucagon
(10
8 M) had similar effects.
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Fig. 9.
Membrane potential. Representative
perforated patch current-clamp recordings of the membrane potential of
a MIN6 cell. A, response to 10 mM glucose;
B, response to 10 9 M miniglucagon
in the continued presence of 10 mM glucose; C,
response to 10
7 M somatostatin in the
continued presence of 10 mM glucose. All three records are
from the same cell. Because of the slow time course of recovery, only
the onset of the responses are illustrated.
7 M somatostatin
produced very similar effects to 10
9 M
miniglucagon: hyperpolarization of the membrane potential and abolition
of the electrical activity. These effects of somatostatin are very
similar to those that have been previously reported for beta cells,
isolated from normal mouse using a similar concentration of peptide
(30, 31).
2 receptors, potassium channels in a G protein-sensitive manner (38), we have
verified that yohimbine, an
2-receptor blocker, did not interfere
with the miniglucagon action. Indeed, 10 mM glucose-induced insulin release by MIN6 cells was inhibited by 0.1 nM
miniglucagon to the same extent in the absence and in the presence of
10 µM yohimbine (data not shown).
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Fig. 10.
Reversal of somatostatin and miniglucagon
inhibition of insulin release from MIN6 cells by pertussis toxin.
MIN6 cells were incubated overnight in culture medium containing 200 ng/ml Bordetella pertussis toxin or in standard culture
medium. Statistical significance was determined by comparing the data
obtained in the presence and in the absence of Bordetella
pertussis toxin. **, p < 0.01; ***, p < 0.001.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
11 M range because glucagon acts on beta
cells, and on other target cells, in the nanomolar range (36). The
threshold effects of miniglucagon observed in the 10
13 to
10
12 M range gives miniglucagon the status of
a possible local regulator of insulin release even if the peptide is
released at a much lower rate than glucagon. Further studies are
required, however, to analyze the conditions under which miniglucagon
is released from the alpha cells to get further insight in the precise
role of miniglucagon in islet physiology.
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FOOTNOTES |
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* 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.
§ Recipient of Wellcome Trust Career Development award No. 042345.
¶ To whom correspondence should be addressed. Tel.: +33 4 67 41 52 20; Fax: +33 4 67 41 52 22; E-mail: bataille{at}u376.montp.inserm.fr.
2 P. Blache and M. Dufour, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: MGE, miniglucagon-generating endopeptidase; tGLP-1, glucagon-like peptide-1-(7-36) amide; BSA, bovine serum albumin; IBMX, 3-isobutyl-1-methylxanthine; VDCC, voltage-dependent Ca2+ channels; InsP3, inositol trisphosphate.
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REFERENCES |
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