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INTRODUCTION |
Glucose-induced insulin secretion from pancreatic
cells is
regulated by generation of ATP through glucose metabolism and an
increase in the cytosolic calcium concentration
([Ca2+]c)1
(1-4). The following cascade has been generally accepted as the
glucose-induced insulin secretory pathway. When glucose is metabolized
in the cytosol and mitochondria, ATP is generated to promote closure of
ATP-sensitive potassium (KATP) channels, and this
depolarizes the plasma membrane potential. The depolarization of the
plasma membrane leads to activation of voltage-dependent Ca2+ channels, with subsequent Ca2+ entry into
the cytosol. The rise in [Ca2+]c is thought to
finally trigger exocytosis of insulin from secretory vesicles (1-3).
Thus, inhibitors of glucose metabolism (5) and Ca2+
channel-blocking agents, such as verapamil (6), nifedipine (7), and
divalent cations (8-14), suppress glucose-induced insulin secretion.
Moreover, recent studies showed that the intramitochondrial Ca2+ concentration ([Ca2+]m)
increases in accordance with the rise in [Ca2+]c
upon glucose stimulation via a calcium transporter on the inner
mitochondrial membrane (calcium uniporter) (15, 16) and that the
increase in [Ca2+]m is closely associated with
glucose-induced insulin secretion (17, 18).
To examine the involvement of [Ca2+]m in
glucose-induced insulin secretion from isolated islets, we tested the
effects of ruthenium red and hexamminecobalt(III) (HAC), both of which have been reported to be inhibitors of the mitochondrial calcium uniporter (15, 18-20), on the secretion. Ruthenium red was also reported to inhibit insulin secretion from permeabilized
cells through inhibition of the calcium uniporter (18). We also observed that
ruthenium red at 100 µM severely suppressed
glucose-induced insulin secretion in mouse islets. However, under these
conditions, Ca2+ influx through the plasma membrane was
almost completely inhibited. These results indicated that ruthenium red
was not a suitable agent to study the relationship of
[Ca2+]m to glucose-induced insulin secretion in
intact cells, different from the case of permeabilized cells. In
contrast, HAC at 2 mM suppressed glucose-induced insulin
secretion to the same extent as 100 µM ruthenium red,
without affecting the increase in [Ca2+]c in
response to glucose. However, different from our initial expectations,
HAC failed to affect glucose-stimulated increases in
[Ca2+]m under these conditions. In this study, we
tried to determine which step of the glucose-induced insulin secretory pathway was inhibited by HAC. HAC failed to suppress oxidation of
glucose, glucose-stimulated generation of ATP, or Ca2+
current through the plasma membrane, indicating that the inhibitory effect of HAC on insulin secretion was exerted distal to
[Ca2+]c elevation. Our results suggested that the
dissociation of the SNARE complex formed in the sequence of exocytotic
events was prevented in the presence of HAC. Thus, HAC may serve as a useful probe to monitor steps distal to elevation of
[Ca2+]c in the glucose-stimulated insulin
secretory pathway.
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EXPERIMENTAL PROCEDURES |
Materials--
HAC, chlortetracycline, rhodamine 123, and the
ATP bioluminescent assay kit were purchased from Sigma.
Fura-2/acetoxymethyl ester was from Molecular Probes, Inc. (Eugene,
OR). D-[6-14C]Glucose was from American
Radiolabeled Chemicals (St. Louis, MO). Solvable was from Packard
Instrument Co. The insulin radioimmunoassay kit, cAMP enzyme-linked
immunoassay kit, and ECL protein detection kit were from Amersham
Pharmacia Biotech (Uppsala, Sweden). Anti-syntaxin and
anti-vesicle-associated membrane protein (VAMP) antibodies were from
Upstate Biotechnology, Inc. (Lake Placid, NY). Nitrocellulose membranes
were from Schleicher & Schüll (Dassel, Germany).
Preparation of Islets--
Islets were isolated by collagenase
digestion and manual picking from the pancreases of 10-16-week-old
male C57BL/6N mice (21). These islets were used for the experiments
immediately after isolation.
Cell Culture--
HC9 cells were maintained as described
elsewhere (22). They were cultured in Dulbecco's modified Eagle's
medium containing 25 mmol/liter glucose, 1 mmol/liter pyruvate, 15%
horse serum, 2.5% fetal bovine serum, 100 µg/ml streptomycin, and
100 units/ml penicillin at 37 °C in a 95% air and 5%
CO2 atmosphere. In the course of experiments, cells were
maintained with one passage/week.
Insulin Secretion from Islets--
Insulin release from
pancreatic islets was measured in static incubation or perifusion
incubation with Krebs-Ringer bicarbonate (KRB) buffer composed of 129 mM NaCl, 4.8 mM KCl, 1.2 mM
MgSO4, 1.2 mM KH2PO4,
2.5 mM CaCl2, 5 mM
NaHCO3, 0.2% bovine serum albumin, and 10 mM
HEPES (pH 7.4) (4). HAC was present, if used, throughout the
experiment. In static incubation experiments, batches of 10 freshly
isolated islets were preincubated at 37 °C for 20 min in KRB buffer
containing 2.8 mM glucose. The preincubation solutions were
replaced with KRB buffer containing test agents, and batches of islets
were incubated at 37 °C for 60 min. Insulin released in these
supernatants was measured by radioimmunoassay. In perifusion incubation, 30 freshly isolated islets were suspended in 500 µl of
Bio-Gel G-10 beads in each perifusion chamber and perifused with KRB
buffer at 37 °C at a flow rate of 0.6 ml/min. Islets were perifused
for 30 min in the presence of 2.8 mM glucose prior to
stimulation with 16.7 mM glucose. Perifusate fractions were collected, and insulin in these samples was measured by radioimmunoassay.
Insulin Secretion from
HC9 Cells--
Insulin release from
HC9 cells was measured in static incubation experiments (23).
HC9
cells grown in 60-mm diameter wells were preincubated at 37 °C for
20 min in KRB buffer containing 0.1 mM glucose. The
preincubation solutions were replaced with KRB buffer containing test
agents, and the cells were incubated at 37 °C for 60 min. HAC was
present, if used, throughout the experiment. Insulin released in these
incubation media was measured by radioimmunoassay.
D-[6-14C]Glucose
Oxidation--
Glucose oxidation in islets was measured by the
generation of 14CO2 from
D-[6-14C]glucose (24). Batches of 10 freshly
isolated islets were incubated at 37 °C for 90 min in KRB buffer
containing the indicated concentrations of glucose and HAC. Total
radioactivity added to KRB buffer was 150 kBq/ml for
D-[6-14C]glucose, resulting in specific
activities of 16-125 Bq/nmol. The 14CO2
produced was made volatile by adding HCl, captured by Solvable, and
measured by liquid scintillation counting.
Measurement of Fluorescence--
For measurement of fluorescence
parameters, a single islet was placed under a microscope (IMT-2,
Olympus, Tokyo, Japan) and perifused with Sol II buffer
composed of 150 mM NaCl, 5 mM KCl, 1.0 mM MgCl2, 2.0 mM CaCl2,
and 10 mM HEPES (pH 7.4) at 37 °C (4). Fluorescence was
excited with light emitted from a xenon lamp (TILL Photonics),
collected through interference filters (Olympus), and detected using a
photomultiplier (NT5783, Hamamatsu Photonics, Hamamatsu, Japan).
The autofluorescence of the reduced forms of NAD and NADP, referred to
as NAD(P)H, was excited at 360 nm and filtered at 470 nm (4).
For measurement of mitochondrial inner membrane potential, islets were
loaded with 10 µg/ml rhodamine 123 at 37 °C for 10 min. Rhodamine
123 fluorescence was excited at 490 nm and filtered at 530 nm (18). For
monitoring of [Ca2+]c, islets were loaded with 15 µM fura-2/acetoxymethyl ester at 37 °C for 1 h.
The fluorescence was excited in a dual-wavelength ratiometric
mode at 340 and 380 nm. The emission wavelength was filtered at 500 nm.
[Ca2+]c was expressed as the 340/380 nm emission
ratio (5). For measurement of [Ca2+]m, islets
were perifused with Sol II buffer containing 50 µM
chlortetracycline. Chlortetracycline fluorescence was excited at 400 nm
and filtered at 560 nm (25). Since the base-line fluorescence of
chlortetracycline steadily increased with time, the increase was
subtracted from the raw data.
Contents of ATP and cAMP--
The contents of ATP and cAMP in
whole islets were determined as described previously (26, 27). Batches
of 10 islets were incubated at 37 °C for 60 min in KRB buffer
containing the indicated concentrations of glucose and HAC. The
incubation was stopped by addition of ice-cold HClO4, and
islets were homogenized by sonication. The lysates were neutralized by
addition of NaOH. ATP and cAMP contents in the supernatant were
measured using a bioluminescent assay kit and an enzyme-linked
immunoassay kit, respectively.
Effect of HAC on the SNARE Complex in
HC9 Cells--
SNARE
complexes formed in
HC9 cells were detected by a
co-immunoprecipitation method (23).
HC9 cells were preincubated at
37 °C for 30 min in KRB buffer containing 0.1 mM
glucose. The medium used for preincubation was replaced with fresh KRB
buffer containing test agents, and incubations were carried out at
37 °C for 5 min. After incubation, cells were washed with cold
phosphate-buffered saline and incubated in 700 µl of lysis buffer (50 mM NaCl, 15.7 mM
NaH2PO4, 1.47 mM
KH2PO4, 2.68 mM KCl, 1% Nonidet
P-40, 1 mM dithiothreitol, 15 mM
MgCl2, 100 µg/ml aprotinin, 100 µg/ml leupeptin, and
100 µg/ml phenylmethylsulfonyl fluoride (pH 7.4)) at 4 °C for 30 min. The samples were collected and centrifuged at 16,000 × g for 5 min. The supernatants were collected and used for immunoprecipitation.
Immunoprecipitation and Immunoblotting--
SNARE complexes in
the lysates of
HC9 cells were detected by a co-immunoprecipitation
method (23). Samples were incubated with anti-syntaxin antibody and
protein G-agarose or with anti-VAMP antibody and anti-mouse IgM-agarose
for 2 h at 4 °C. The immune complexes precipitated with
agarose beads were boiled in Laemmli sample buffer for 5 min (28). All
samples were separated on 12% SDS-polyacrylamide gels; transferred to
polyvinylidene fluoride membranes; and probed with anti-VAMP or
anti-syntaxin antibody, followed by detection with the ECL kit.
Statistical Analysis--
Statistical analysis was performed
using Student's t test for unpaired comparisons. Values are
shown as means ± S.E.
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RESULTS |
Effect of HAC on Glucose-induced Insulin Secretion--
The effect
of HAC on glucose-induced insulin secretion from isolated islets was
measured in static incubations for 60 min (Fig.
1A). In the presence of 2.8 mM glucose, islets released 0.5 ± 0.1 ng of
insulin/h/islet (n = 4), and HAC at 2 mM
failed to exert significant inhibition of insulin secretion under these conditions (0.4 ± 0.1 ng/h/islet, n = 4). In the
presence of 22.2 mM glucose, insulin secretion from
isolated islets increased to 3.2 ± 0.1 ng/h/islet
(n = 4), and HAC inhibited this secretion in a
dose-dependent manner. Thus, the inhibition by 0.1, 0.5, 1, and 2 mM HAC amounted to 0, 50, 71, and 90% of the
glucose-stimulated increase in insulin secretion, respectively.

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Fig. 1.
Inhibitory effect of HAC on glucose-induced
insulin secretion from islets. A, glucose-induced
insulin secretion from islets in static incubations. Batches of 10 freshly isolated islets were incubated in KRB buffer with the indicated
concentrations of glucose and HAC at 37 °C for 60 min after a 20-min
preincubation in the presence of 2.8 mM glucose. HAC was
present, if used, throughout the experiment. The results are expressed
as means ± S.E. (n = 4). B,
glucose-induced insulin secretion from islets in perifusion
incubations. Each perifusion chamber contained 30 freshly
isolated mouse islets. Islets were perifused for 30 min at a flow rate
of 0.6 ml/min in the presence of 2.8 mM glucose. The
glucose concentration was then raised to 16.7 mM at 0 min.
HAC (2 mM) was absent ( ), present ( ), or removed at
20 min ( ). Experiments were carried out at 37 °C; and HAC was
present, if used, from the preincubation time. The results are
expressed as means ± S.E. (n = 3).
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Perifusion experiments revealed the effect of HAC on the kinetics of
glucose-induced insulin secretion from isolated islets (Fig.
1B). An increase in glucose concentration from 2.8 to 16.7 mM induced a biphasic insulin secretion in control islets
(Fig. 1B,
). In the presence of 2 mM HAC, the
first phase of insulin secretion (0-10 min) over the basal values was
inhibited by 80%, and the second phase was completely abrogated (Fig.
1B,
). Removal of HAC from the perifusate at 20 min
caused a prompt and full restoration of insulin secretion with a
transient overshooting above the second phase level of the control
(Fig. 1B,
).
Effect of HAC on Mitochondrial Metabolism and ATP
Generation--
To examine the effect of HAC on mitochondrial
metabolism eventually leading to ATP generation, we measured
glucose-induced changes in D-[6-14C]glucose
oxidation, the level of NAD(P)H, mitochondrial inner membrane
potential, and ATP content in islets. Oxidation of
D-[6-14C]glucose to
14CO2, which well reflects the tricarboxylic
acid cycle activity at the level of isocitrate dehydrogenase and
-ketoglutarate dehydrogenase (24), was increased by 13-fold when
extracellular glucose was raised from 2.8 to 22.2 mM in
control islets (0.6 ± 0.1 and 8.5 ± 1.5 pmol/h/islet,
respectively; n = 4) (Table
I). HAC at 2 mM did not
affect D-[6-14C]glucose oxidation in the
presence of 2.8 mM glucose (0.6 ± 0.2 pmol/h/islet,
n = 4). In the presence of 22.2 mM glucose,
HAC also failed to significantly change
D-[6-14C]glucose oxidation (11.5 ± 1.8 pmol/h/islet, n = 4) compared with control islets
incubated at the same glucose concentration.
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Table I
Effects of HAC on D-[6-14C]glucose oxidation and
ATP generation in islets
Glucose oxidation was measured by generation of 14CO2
from D-[6-14C]glucose. Batches of 10 mouse islets
were incubated at 37 °C for 90 min in KRB buffer containing the
indicated concentrations of glucose with or without 2 mM
HAC. The 14CO2 produced was made volatile by adding
HCl, captured by Solvable, and measured by liquid scintillation
counting. The ATP content of islets was measured after incubation at
37 °C for 60 min in KRB buffer containing the indicated
concentrations of glucose with or without 2 mM HAC. Batches
of 10 islets were used for each condition. The incubation was stopped
by addition of ice-cold HClO4, and extracts from islet
homogenates were neutralized by addition of NaOH. ATP contents in these
lysates were measured by a luminometric method. The results are
expressed as means ± S.E. (n = 4).
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We then monitored the autofluorescence of NAD(P)H (Fig.
2A), which mainly reflects
that of the mitochondrial NADH pool (29). The autofluorescence of
NAD(P)H was increased above basal levels by 22.2 mM glucose
in control islets (396 ± 49 arbitrary units, n = 4). In the presence of 2 mM HAC, although the increase to a
plateau in autofluorescence was delayed by 3 min, the magnitude of the
increase at 10 min after glucose stimulation (393 ± 71 arbitrary
units, n = 4) was not significantly different from that observed in control islets. Furthermore, we measured glucose-stimulated hyperpolarization of the mitochondrial membrane, which is formed by the
respiratory chain function and in turn drives ATP synthesis, with the
fluorescence of rhodamine 123 (Fig. 2B). In control islets, the fluorescence of rhodamine 123 was decreased in response to glucose
stimulation (578 ± 38 arbitrary units, n = 4),
which reflects the shift in the mitochondrial membrane potential to
hyperpolarization (18). HAC at 2 mM failed to cause any
significant change in glucose-induced hyperpolarization of the
mitochondrial membrane (565 ± 10 units, n = 4).

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Fig. 2.
Effect of HAC on glucose-induced changes in
metabolic parameters and cytosolic and mitochondrial calcium
concentrations in islets. The glucose concentration was raised
from 2.8 to 22.2 mM at 0 min. HAC was present, if used, at
2 mM during the preincubation period to the end of the
experiment. The traces shown are representative of at least
four experiments. A, representative time course of changes
in the level of NAD(P)H induced by 22.2 mM glucose. The
NAD(P)H level was measured at 470 nm, with its autofluorescence excited
at 360 nm. B, representative time course of changes in the
mitochondrial membrane potential induced by 22.2 mM
glucose. The mitochondrial inner membrane potential was measured with
rhodamine 123 (Rh123), the fluorescence of which was excited
at 490 nm and filtered at 530 nm. C, representative time
course of changes in [Ca2+]c induced by 22.2 mM glucose. [Ca2+]c was measured with
fura-2, the fluorescence of which was excited in a dual-wavelength
ratiometric mode at 340 and 380 nm. The emission wavelength was
filtered at 500 nm. [Ca2+]c is expressed as the
340/380 nm ratio. D, representative time course of changes
in [Ca2+]m induced by 22.2 mM
glucose. The changes in [Ca2+]m were monitored
with chlortetracycline (CTC), the fluorescence of which was
excited at 400 nm and filtered at 560 nm.
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Next, we measured the ATP content of islets to directly monitor the
efficiency of mitochondrial ATP synthesis. After a 1-h static
incubation with 22.2 mM glucose at 37 °C, the ATP
content was increased by 40% (12.0 ± 0.8 pmol/islet,
n = 4) compared with that with 2.8 mM
glucose in control islets (8.5 ± 0.4 pmol/islet, n = 4) (Table I). When 2 mM HAC was added,
the ATP content of islets incubated with 2.8 mM glucose
remained unchanged (9.1 ± 0.5 pmol/islet, n = 4),
whereas a slight, but not significant increase in ATP content over the
control islets was noted in the presence of 22.2 mM glucose
(14.5 ± 1.0 pmol/islet, n = 4). These results
indicated that HAC has no inhibitory effects on ATP generation through
mitochondrial glucose metabolism.
Effect of HAC on the Glucose-induced Rise in Cytosolic and
Mitochondrial Ca2+ Concentrations--
To investigate the
effect of HAC on the increase in [Ca2+]c in
response to glucose stimulation, we monitored the fluorescence of
fura-2/acetoxymethyl ester excited at 340 and 380 nm in perifused islets (Fig. 2C). When the glucose concentration was
increased from 2.8 to 22.2 mM,
[Ca2+]c, which was expressed as the 340/380 nm
fluorescence ratio, was increased in control islets by 0.143 ± 0.023 (n = 5). In the presence of 2 mM HAC,
[Ca2+]c also rose in response to glucose
stimulation (0.123 ± 0.018, n = 6;
p = 0.51 compared with control islets), although the
time taken to increase to a plateau was delayed by 1 min compared with
the controls. Moreover, the duration (2.6 ± 0.1 min,
n = 6) and magnitude (0.024 ± 0.002, n = 6) of "phase 0" [Ca2+]c
change below a base-line level in islets treated with HAC were not
different from those of controls (duration, 2.8 ± 0.1 min,
n = 5; magnitude, 0.022 ± 0.003, n = 5). We also directly measured the plasma membrane
potential and Ca2+ currents of single
cells with patch
clamp techniques (30, 31) and found that HAC at 2 mM did
not interfere with 22.2 mM glucose-responsive changes in
this potential or currents (data not shown).
It was previously reported that HAC inhibits the mitochondrial calcium
uniporter, which mediates Ca2+ entry into the mitochondrial
matrix in response to changes in the mitochondrial membrane potential
(15, 16). It was also reported that [Ca2+]m,
which rises in accordance with [Ca2+]c upon
glucose stimulation, is associated with glucose-induced insulin
secretion (17). We thus measured [Ca2+]m with
chlortetracycline. Chlortetracycline is a fluorescent Ca2+
probe that allows continuous monitoring of an intramitochondrial Ca2+ pool bound to the inner mitochondrial membrane,
possessing a substantial fluorescence intensity suitable for
quantitative measurement (Fig. 2D) (25). In control islets,
chlortetracycline fluorescence was increased above the basal level by
233 ± 17 arbitrary units (n = 8) in response to
22.2 mM glucose. In the presence of 2 mM HAC,
an increase in chlortetracycline fluorescence in response to glucose
(234 ± 17 arbitrary units, n = 7) was also
observed and was not significantly different in magnitude from that of control islets. However, the onset of the increase was delayed by 1 min
compared with controls.
Effect of HAC on KCl- or Mastoparan-stimulated Insulin
Secretion--
A high concentration of extracellular KCl decreases
K+ conductance and leads to depolarization of the plasma
membrane, with subsequent activation of voltage-dependent
Ca2+ channels (32). This effect of KCl causes elevation of
[Ca2+]c and results in insulin secretion by
cells. We examined whether HAC also inhibits this
depolarization-induced insulin secretion, which does not require
mitochondrial glucose metabolism or ATP synthesis. HAC did not affect
insulin secretion at the basal concentration of KCl (4.8 mM). When 50 mM KCl was added to the medium,
insulin secretion was increased to 3.6 ± 0.6 ng/h/islet (n = 4). However, 2 mM HAC decreased
KCl-stimulated insulin secretion to 1.3 ± 0.1 ng/h/islet
(n = 4) (Fig.
3A).

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Fig. 3.
Effect of HAC on KCl- or
mastoparan-stimulated insulin secretion from islets. A,
KCl-stimulated insulin secretion from islets. Batches of 10 freshly
isolated islets were incubated in KRB buffer containing the indicated
concentrations of glucose, KCl, and HAC at 37 °C for 60 min after
preincubation for 20 min in the presence of 2.8 mM glucose
and 4.8 mM KCl. HAC was present, if used, throughout the
experiments. The results are expressed as means ± S.E.
(n = 4). B, mastoparan-stimulated insulin
secretion from islets in the absence of extracellular Ca2+.
Batches of 10 freshly isolated islets were incubated in KRB buffer
containing the indicated concentrations of glucose, mastoparan, and HAC
at 37 °C for 60 min after preincubation for 20 min in the presence
of 2.8 mM glucose. Extracellular Ca2+ was
absent; and HAC was present, if used, throughout the experiments. The
results are expressed as means ± S.E. (n = 4).
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Mastoparan, a tetradecapeptide purified from wasp venom, activates
heterotrimeric G-proteins and stimulates insulin secretion from
cells even in the absence of extracellular Ca2+ (33, 34).
We examined the effect of HAC on mastoparan-stimulated insulin
secretion in the absence of extracellular Ca2+ (Fig.
3B). Mastoparan at 10 µM increased insulin
secretion to 3.5 ± 0.4 ng/h/islet (n = 4) in the
absence of external Ca2+, whereas 22.2 mM
glucose elicited no significant increase (0.6 ± 0.1 ng/h/islet,
n = 4) under these Ca2+-free conditions.
HAC at 2 mM inhibited mastoparan-stimulated insulin
secretion by 80%.
Effect of HAC on cAMP Generation--
An increase in cAMP, which
is synthesized from ATP by adenylyl cyclases and eliminated by
phosphodiesterases, potentiates glucose-stimulated insulin secretion
through the activation of cAMP-dependent protein kinases
(35). In control islets, the cAMP contents were 45.7 ± 4.5 fmol/islet (n = 4) at 2.8 mM glucose and
49.3 ± 3.0 fmol/islet (n = 4) at 22.2 mM glucose. The nonselective phosphodiesterase inhibitor
3-isobutyl-1-methylxanthine (IBMX) at 1 mM elevated cAMP
levels to 611.7 ± 18.7 fmol/islet (n = 4) at 2.8 mM glucose and to 837.8 ± 58.2 fmol/islet
(n = 4) at 22.2 mM glucose (Fig.
4A). However, IBMX potentiated
insulin secretion (21.5 ± 1.3 ng/h/islet, n = 4)
only in the presence of 22.2 mM glucose (Fig.
4B). In the presence of HAC at 2 mM, IBMX also
elevated cAMP levels at both 2.8 and 22.2 mM glucose
(755.5 ± 54.1 and 727.1 ± 27.3 fmol/islet, respectively;
n = 4) (Fig. 4A). However, IBMX-mediated
potentiation of insulin secretion in response to 22.2 mM
glucose was virtually negated to 4.0 ± 0.5 ng/h/islet (n = 4) in the presence 2 mM HAC (Fig.
4B).

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Fig. 4.
Effect of HAC on the generation of cAMP and
the potentiation of glucose-induced insulin secretion by cAMP in
islets. Batches of 10 islets were incubated at 37 °C for 60 min
in KRB buffer containing the indicated concentrations of glucose, IBMX,
and HAC. The incubation was stopped by addition of ice-cold
HClO4 after separation of supernatants for measurement of
secreted insulin (B). The extracts from islet homogenates
were neutralized by addition of NaOH. The contents of cAMP in these
lysates (A) were measured by enzyme-linked immunoassay. The
results are expressed as means ± S.E. (n = 4).
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Effect of HAC on the Exocytosis Machinery--
To examine whether
HAC affects the formation or dissociation of the exocytosis machinery,
we performed a co-immunoprecipitation study using anti-syntaxin and
anti-VAMP antibodies (23). Because of difficulties in isolating the
large amounts of pancreatic
cells needed to detect the complex of
SNARE proteins (data not shown), we used mouse insulin-secreting
HC9
cells, which preserve the characteristics of progenitor mouse islets
(22). Insulin secretion from
HC9 cells was increased ~5-fold in
response to 22.2 mM glucose compared with the basal
secretion level in the presence of 0.1 mM glucose, and this
glucose-induced increase in insulin secretion was inhibited by 50% in
the presence of 2 mM HAC (Fig.
5A). In the absence of HAC,
VAMP co-immunoprecipitated with syntaxin (Fig. 5B,
first lane) and syntaxin co-immunoprecipitated with VAMP
(Fig. 5C, first lane) were clearly detectable
under the basal glucose conditions, and both were significantly
decreased to 54 and 28% of the original levels, respectively, by 22.2 mM glucose stimulation. In the presence of HAC at 2 mM, the amounts of co-immunoprecipitated VAMP (Fig.
5B, third lane) and syntaxin (Fig. 5C,
third lane) were not affected at the basal glucose
concentration compared with those in the absence of HAC. These results
indicated that the SNARE complex in the docked state was retained even
in the presence of HAC under basal glucose conditions. However, under stimulated conditions with 22.2 mM glucose, HAC suppressed
the decrease in co-immunoprecipitated VAMP (Fig. 5B,
fourth lane) and syntaxin (Fig. 5C, fourth
lane), so these two proteins remained to form the complex, as if
they became stabilized by HAC. In each lane, the amounts of VAMP
protein detected by Western blotting without immunoprecipitation were
constant (data not shown), indicating that HAC did not induce a
decrease in or degradation of proteins.

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Fig. 5.
Effect of HAC on the SNARE complex in whole
cells. A, glucose-induced insulin secretion from
HC9 cells. HC9 cells were incubated in KRB buffer with the
indicated concentrations of glucose and HAC at 37 °C for 60 min
after a 20-min preincubation in the presence of 0.1 mM
glucose. HAC was present, if used, throughout the experiments. Insulin
secreted in the media was measured by radioimmunoassay. The results are
expressed as means ± S.E. (n = 4). B
and C, detection of the SNARE complex by the
co-immunoprecipitation method. HC9 cells were incubated for 5 min
with the indicated concentrations of glucose and HAC subsequent to a
30-min preincubation with 0.1 mM glucose at 37 °C. HAC
(2 mM) was present, if used, during the preincubation
period to the end of the incubation. The lysates obtained from these
cells were used for immunoprecipitation (IP) with
anti-syntaxin (B) or anti-VAMP (C) antibody. The
precipitated samples were boiled in Laemmli sample buffer; separated on
12% SDS-polyacrylamide gels; transferred to polyvinylidene difluoride
membranes; and probed with anti-VAMP (B) or anti-syntaxin
(C) antibody, followed by detection with ECL. The relative
intensities of immunoprecipitated proteins on the Western blot
(WB) membranes, compared with controls at 0.1 mM
glucose without HAC, are shown beneath the representative blots
(B, means ± range, n = 2;
C, means ± S.E., n = 3).
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DISCUSSION |
HAC ([Co(NH3)6]3+) is a
trivalent complex cation of Co(III) and amine that has been known and
used as an inhibitor of the mitochondrial calcium uniporter (15, 20)
and a radiation-sensitizing agent (36). The importance of
[Ca2+]m in regulation of glucose-induced insulin
secretion from pancreatic
cells (18) prompted us to study the
effect of HAC on the secretory pathway. We found that HAC strongly
inhibited glucose-induced insulin secretion and designed the present
experiments to further evaluate the mechanism of HAC inhibition of
insulin secretion using different types of insulin secretagogues. HAC inhibited glucose-induced insulin secretion from pancreatic islets without affecting Ca2+ uptake into mitochondria. This
inhibitory effect of HAC on the secretion was acutely and completely
reversed with a transient overshooting by removing HAC from the
perifusate. This indicated that the insulin secretory mechanisms had
not been irreversibly altered by HAC. It is noteworthy that HAC showed
practically no inhibition of basal insulin secretion, indicating that
the inhibitory effects of HAC are exerted at sites specific to
stimulated secretion. Features similar to the inhibitory and withdrawal
effects of HAC on glucose-induced insulin secretion have been observed
in the case of inhibition by several divalent cations, including
Co2+, Zn2+, Ni2+, Mn2+,
and Mg2+ (8-13). The inhibitory effects of these divalent
cations are exerted mainly on stimulated insulin secretion (8, 9,
11-13). In particular, the inhibitory effect of Co2+,
Mg2+, and Mn2+ on glucose-induced insulin
secretion is reversed with a transient overshooting by removal of these
cations from the perifusate (8, 12, 13). Moreover, the inhibitory
effects of Co2+, Zn2+, and Ni2+ on
insulin secretion show no parallel effects on the oxidation of glucose
(8, 9, 11), resembling the effect of HAC. However, each of these
cations is known to be a blocker of Ca2+ channels and has
been reported to inhibit glucose-stimulated calcium uptake into
cells (8, 12, 13). Furthermore, Co2+ and Zn2+
have been reported to inhibit the electrical activities of the plasma
membrane induced by glucose (10). It is generally accepted that a rise
in the cytosolic free Ca2+ concentration is essential for
insulin secretion from
cells (1). Thus, with most of these divalent
cations, interference with Ca2+ influx from the
extracellular space is the main cause of suppression of insulin
secretion (8, 10, 12, 13). In contrast to these divalent cations, HAC
failed to suppress glucose-stimulated Ca2+ influx and the
plasma membrane electrical activities. These results suggest that HAC
inhibits insulin secretion at a site(s) distal to the elevation of
[Ca2+]c. The inhibitory effect of HAC on
depolarization-induced insulin secretion also validates this
localization. We also examined whether HAC affects the generation and
action of cAMP, which is one of the major second messengers and has
been reported to potentiate insulin secretion via its action on the
KATP channel-independent pathway (37). HAC clearly
suppressed the potentiation of glucose-induced insulin secretion by
cAMP without inhibiting its generation.
The suppressive effect of HAC on mastoparan-stimulated insulin
secretion in the absence of extracellular Ca2+ provides a
clue to the mechanism of HAC inhibition of insulin secretion.
Mastoparan is thought to directly stimulate insulin secretion from
cells by activation of exocytosis-linked heterotrimeric guanosine
triphosphate-binding proteins (Ge) without employing influx
of Ca2+ through the plasma membrane (33, 38). As in
neurotransmitter release, the molecular machinery of exocytosis for
regulated insulin secretion involves SNAREs, which have been highly
conserved from yeast to mammals (39). SNAREs have been identified as
VAMP on the vesicular membrane and as SNAP-25
(synaptosome-associated protein of 25 kDa) and syntaxin on the plasma
membrane (40-45). SNAREs form a very stable core complex called the
SNARE complex in the presence of Ca2+ (46), and this
complex is dissociated by the action of NSF (N-ethylmaleimide-sensitive
fusion protein) and soluble NSF attachment proteins to
mediate a membrane fusion process (47-49). In pancreatic
cells, it
has been shown that the SNARE complexes are disrupted through the
insulin secretion process (23) and that the specific cleavage of SNAREs
by botulinum toxins inhibits Ca2+-dependent
insulin release (40, 42, 43), in which Ca2+ is thought to
mediate exocytosis through synaptotagmin, which, depending on the
concentration of free cytosolic Ca2+, interacts with
phospholipids and syntaxin (50). On the other hand, Ge is
thought to be directly coupled with exocytosis in a number of cell
types and to stimulate exocytosis independently of any elevation of
[Ca2+]c (33, 38). Moreover, an interaction
between heterotrimeric G-proteins and syntaxin was demonstrated in a
recent study (51). HAC was demonstrated to inhibit both
Ca2+-dependent and Ca2+-independent
insulin secretion. These results are compatible with a concept that the
inhibitory effect of HAC is exerted on the exocytotic process per
se, or at sites closely proximal to this, within the final
processes of stimulus-secretion coupling.
The results of a co-immunoprecipitation study of the SNARE complex
demonstrated that the complex was clearly detectable under basal
glucose conditions even in the presence of HAC, but that the disruption
of the complex triggered by glucose stimulation was prevented by HAC
(Fig. 5). A recent study reported that the co-immunoprecipitated SNARE
complex reflects a physiologically releasable pool of docked
insulin-containing granules and is decreased during the early phase of
glucose-stimulated insulin release (23). Thus, the results of our
co-immunoprecipitation study indicated that HAC essentially failed to
inhibit the docking of granules with the plasma membrane and that HAC
exerted its inhibitory effect after the docking step in the exocytosis
machinery. This finding narrows the possible sites of HAC action
directly to the disruption of the SNARE complex per se or to
the cascade that triggers the disruption. In this regard, the molecules
interacting with HAC might be key components of the exocytotic
machinery, and identification of these molecules should be a focus of
future investigations. In addition to the SNARE complex molecules, the
secretory vesicle-acidifying machinery might be among these candidate
molecules since glutamate, generated through
-ketoglutarate from
glucose, was reported to be a metabolic signal that augments
fuel-stimulated insulin secretion via its action on secretory vesicle
acidification (52), although the relationship between the fusing and
acidifying machineries of
cells has not been elucidated.
In conclusion, we have found that HAC inhibits glucose-induced insulin
secretion from isolated pancreatic islets without inhibiting glucose
metabolism and Ca2+ influx into the cytosol. Furthermore,
HAC also extends its inhibitory effect to mastoparan-stimulated insulin
secretion in the absence of extracellular Ca2+. Although
the sites on which HAC exerts its inhibitory effect have not been
completely elucidated, this compound is likely to affect exocytosis
per se and/or triggers of exocytosis in a reversible manner.
Botulinum toxins, which cleave SNAREs irreversibly, have been the only
agents reported to inhibit the exocytotic machinery to date, except for
antibodies against proteins in the SNARE complex. Therefore, HAC may
potentially serve as a powerful tool to study the exocytotic processes
in
cells. It is also expected that further experiments on the
mechanism of the inhibitory action of HAC will clarify new aspects of
the exocytotic machinery in numerous other tissues such as neuronal synapses.