From the Department of Biochemistry, Faculty of
Pharmaceutical Sciences, Okayama University, Okayama 700-8530, the
** Department of Cell Biology, Institute for Molecular and
Cellular Regulation, Gunma University, Maebashi 371-8512, and the
Department of Physiology, Kansai Medical
University, Moriguchi, Osaka 570-8506, Japan
Received for publication, July 8, 2002, and in revised form, October 21, 2002
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ABSTRACT |
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L-Glutamate is believed to
function as an intercellular transmitter in the islets of Langerhans.
However, critical issues, i.e. where, when and how
L-glutamate appears, and what happens upon stimulation of
glutamate receptors in the islets, remain unresolved. Vesicular
glutamate transporter 2 (VGLUT2), an isoform of the vesicular glutamate
transporter essential for neuronal storage of L-glutamate,
is expressed in L-Glutamate is the major excitatory neurotransmitter
in the central nervous system and plays important roles in
many neuronal processes such as fast synaptic transmission and neuronal
plasticity (1, 2). To use L-glutamate as an intercellular
signaling molecule, neuronal cells develop the glutamatergic system.
Thus, L-glutamate is accumulated in synaptic vesicles
through vesicular glutamate transporters
(VGLUTs),1 and is secreted
through exocytosis. The released L-glutamate binds to the
receptor so as to transmit signals intercellularly. The excess amount
of L-glutamate in synaptic cleft is sequestrated through
plasma membrane-type glutamate transporter.
Recent evidence has indicated that peripheral non-neuronal tissues also
possess the glutamatergic system and use L-glutamate as an
intercellular transmitter (3). The islet of Langerhans, a pancreatic
miniature organ for the hormones regulating the blood glucose level, is
composed of four major types of endocrine cells, i.e.
insulin-secreting Recent findings indicate that brain-specific
Na+-dependent inorganic phosphate cotransporter
(12) and its isoform, differentiation-associated Na+-dependent inorganic phosphate cotransporter
(13), function as VGLUTs and are thus abbreviated as VGLUT1 and VGLUT2,
respectively (14-21). These VGLUTs seem to be potential probes for the
site of L-glutamate release in peripheral tissues as well
as the central nervous system since these transporters are essential
for L-glutamate signal output. We have shown that VGLUT2 is
expressed in During course of the study, we noticed that the expression and
subcellular localization of VGLUTs are of extraordinary significance as
to islet physiology. Here we show that both VGLUT1 and VGLUT2 are
specifically localized with glucagon-containing secretory granules in
Preparations--
Islets of Langerhans were isolated from male
Wistar rats at 7-8 postnatal weeks by the collagenase digestion method
combined with discontinuous Ficoll gradient centrifugation (27). Islets were then handpicked and suspended in a bicarbonate-buffered Hanks' solution supplemented with 0.2% bovine serum albumin.
In some experiments, Immunohistochemistry and Immunoelectronmicroscopy--
Indirect
immunofluorescence microscopy was performed as described previously,
using an Olympus FV-300 confocal laser microscope (18). For
immunoelectronmicroscopy, the LR White embedding immunogold method was
used with a slight modification (30). The animals were anesthetized
with ether and then perfused intracardially with saline followed by
0.2% glutaraldehyde and 4% paraformaldehyde in 0.1 M
phosphate buffer (pH 7.4). Then each pancreas was cut into small
pieces, washed with 0.1 M cacodylate buffer (pH 7.4), stained with uranyl acetate for 2 h, dehydrated, and then embedded in LR White for 2 days at Antibodies--
Site-specific rabbit polyclonal antibodies
against rat VGLUT2 were raised as described previously (18). For the
preparation of anti-VGLUT1 antibodies, DNA fragments encoding G506-S560
were amplified by PCR and cloned into the EcoR1 site of
expression vector pGEX3X (Amersham Biosciences) to form
glutathione S-transferase fusion plasmids. After
transformation, the glutathione S-transferase fusion
proteins encoding G506-S560 were purified on a glutathione-Sepharose 4B
column (Amersham Biosciences) and then injected into a rabbit with
complete adjuvant two times with a 2-week interval. The immunological specificity of the VGLUT1 and VGLUT2 antibodies was proven
using VGLUT1 and VGLUT2 expressed in COS7 cells (15, 18). The mouse monoclonal antibodies against synaptophysin and glucagon were from
Progen. The rat monoclonal antibodies against
L-glutamate were from Dia Sorin Inc. The rat monoclonal
antibodies against somatostatin were from Chemi-Con. The guinea pig
polyclonal antibodies against insulin were from Biogenesis Ltd. The
secondary antibodies conjugated with colloidal gold were obtained from
British Biocell International Ltd. Alexa Fluor 568-labeled anti-mouse
IgG and Alexa Fluor 488-labeled goat anti-rabbit IgG were from
Molecular Probes.
Reverse Transcription Polymerase Chain Reaction
(RT-PCR)--
Total RNA extracted from isolated islets (1 µg) was
transcribed into cDNA in a final volume of 20 µl of a reaction
buffer containing 0.2 mM each dNTP, 10 mM
dithiothreitol, 25 pmol of random octamers, and 200 units of Moloney
murine leukemia virus reverse transcriptase (Amersham Biosciences).
After 1 h of incubation at 42 °C, the reaction was terminated
by heating at 90 °C for 5 min. For PCR amplification, the 10-fold
diluted synthesized cDNA solution was added to the reaction buffer
containing 0.6 mM total dNTP (150 µM each
dNTP), 25 pmol of primers and 1.5 units of Ampli Taq-Gold
DNA polymerase (PerkinElmer Life Sciences). Thirty-five temperature
cycles were conducted, as follows: denaturation at 94 °C for 30 s, annealing at 54 °C for 30 s, and extension at 72 °C for 1 min. The amplification products were analyzed by polyacrylamide gel
electrophoresis. The sequences of the oligonucleotides used as primers
were based on the published sequences of a VGLUT1-specific sense
primer, 5'-AGTGAAATGGAAGACGAGGTT-3' (bases 1687-1707) and antisense
primer, 5'-TTCGGCACGAGCTTGAAACT-3' (bases 2002-2021) (12). DNA
sequencing was performed by the chain-termination method.
Western Blotting--
Samples were denatured with SDS sample
buffer containing 1% SDS and 10% Assay for L-Glutamate Release--
Isolated islets
(20 pieces per assay) or cultured cells (4.0 × 106
cells/dish) were washed three times with DMEM and then incubated in
Ringer's solution containing 10 mM HEPES (pH 7.4), 0.2%
bovine serum albumin and glucose at the specified concentrations for 1 h at 37 °C. Then, the islets or cultured cells were
transferred to 350 µl of the above Ringer's solution containing
glucose at the specified concentrations. At the times indicated,
samples (70 µl) were taken and the amount of L-glutamate
was determined by high pressure liquid chromatography on a RESOLVE C18
column (3.9 × 150 mm; Waters Ltd.) and fluorescence detection as
described previously (22). To determine the total
L-glutamate concentration, islets (50 pieces per assay)
were vigorously homogenized in a bicarbonate-buffered Hanks' solution
in the presence of 6% perchloric acid, sonicated for 5 min, and
centrifuged at 25.000 × g for 15 min. Then, the
supernatant was carefully taken, and its L-glutamate concentration was measured by HPLC, it being found to correspond to
102 ± 13 pmol/islet (n = 3).
Assay for GABA Release--
Islets (20 pieces/assay) or cultured
cells (4.0 × 106 cells/dish) were washed three times
with DMEM and then incubated in a Ringer's solution comprising 10 mM HEPES (pH 7.4), 0.2% bovine serum albumin, and glucose
at the specified concentrations for 1 h at 37 °C. Then, islets
or cells were transferred to 350 µl of a Ringer's solution
containing glucose at the specified concentrations. After 30 min of
incubation, samples (70 µl) were taken and the amount of GABA was
determined by high pressure liquid chromatography on a CAPCELL PAK C18
column (2.0 × 250 mm; Sizeido Co., Ltd.) and amperometric
detection (31). When necessary, L-glutamate, AMPA, or
kainate at 0.5 mM each or CNQX at 50 µM was
also included. In the experiments on MIN6 m9 cells, GABA release was
also quantified by measuring radioactive GABA according to Refs. 32 and
33. Both procedures gave essentially the same results. For loading radiolabeled GABA, MIN6 m9 cells (4.0 × 106
cells/dish) were incubated in DMEM containing 0.2% bovine serum albumin, 1 mM aminoxyacetic acid, and 30 nM
[3H]GABA (specific activity, 60 Ci/mmol; NEN) for 2 h and then washed with a Ringer's solution containing 16.7 mM glucose. Then the cells were incubated in a Ringer's
solution containing glucose at the specified concentrations for 1 h and transferred to 2 ml of the same solution for 30 min as described
above. Then the radioactivity in the culture medium and cell lysate was counted.
Intracellular [Ca2+] Measurement and
Other Procedures--
For the analysis of intracellular
[Ca2+], an Argus 20/CA ratio imaging system
(Hamamatsu Photonics Co., Hamamatsu, Japan) was used (22). Cells
were cultured on a thin glass coverslip precoated with
poly-L-lysine (0.12 mm thick and 40 mm in diameter;
8.0 × 105 cells/coverslip). Then the cells were
treated with 5 µM Fura-2 AM (Dojindo Co., Kumamoto,
Japan) for 50 min at 37 °C and washed twice with the same medium.
The cells were perfused with the warmed Ringer's solution or
Ca2+-Ringer's solution. Images were continuously
taken at 37 °C with a silicon-intensified camera (C2741-08;
Hamamatsu Photonics Co.). The velocity of data acquisition for F334
by F380 images was 4 s at a resolution of 256 × 256 pixels
per image. A personal computer with appropriate software (U4469;
Hamamatsu Photonics Co.) was used to control the optical equipment and
then recording and data analysis. The software enabled subtraction of
background fluorescence, pixel-to-pixel division of F334 by F380
images, fitting of the F334/F380 ratios to a [Ca2+]
calibration curve prepared separately, and digital averaging of the
Ca2+ concentration in multiple cells. Glucagon and insulin
were quantified with enzyme immunoassay assay kits obtained from
Amersham Biosciences and Yanaihara, Inc., according to the
manufacturers' manuals.
Localization of VGLUT2 in Glucagon-containing Secretory
Granules--
Expression and Localization of VGLUT1 in Glucagon-containing
Secretory Granules--
To exclude the possibility of the presence of
VGLUT in
Expression of VGLUT1 in
Double labeling for immunoelecronmicroscopy indicated that gold
particles for VGLUT1 were specifically associated with the membranes of
glucagon-containing secretory granules (Fig. 2D). Like
localization of VGLUT2 shown in Fig. 1, D and E,
essentially no gold particles for VGLUT1 were observed in any
organelles, including secretory granules in Co-secretion of L-Glutamate and Glucagon--
The
specific localization of VGLUTs with glucagon-containing secretory
granules means that L-glutamate is co-stored and
co-secreted with glucagon from
We then investigated whether or not the islets co-secrete
L-glutamate with glucagon. To facilitate the secretion of
glucagon, islets or
Low glucose-stimulated and Ca2+-dependent
secretion of L-glutamate and glucagon was also observed in
Next, we investigated whether or not isoproterenol at 1 µM triggers L-glutamate secretion from
isolated islets since the compound is known to trigger glucagon
secretion by way of L-Glutamate Triggers GABA Secretion from
MIN6 m9 cells are subclonal MIN6
It was found that AMPA and kainate as well as L-glutamate
at 500 µM (glutamatergic stimulation) each stimulated
GABA secretion about 1.5-1.9-fold when the cells were first incubated
with 16.7 mM glucose and then transferred to 3.3 mM glucose (Fig. 5). Glutamate-stimulated GABA release was
also observed when the cells were incubated with 3.3 mM
glucose throughout. Such a stimulatory effect was not observed under
high glucose conditions. The glutamatergic stimulation-evoked GABA
secretion was blocked by CNQX, a specific antagonist for AMPA-type
receptors. Taking 1.1 ± 0.1 nmol/106 cells
(n = 4) as a 100% control of kainate-stimulated GABA
release, the omission of Ca2+ upon incubation of the cells
with EGTA-AM at 50 µM inhibited the glutamatergic
stimulation-evoked GABA release by 93 ± 4.4% (n = 4). Nifedipine, an L-type voltage-gated Ca2+
channel blocker, at 20 µM inhibited the kainate-evoked
GABA release by 70 ± 11% (n = 4). When
Na+ in the medium was replaced with
N-methyl-D-glucamine, kainaite-evoked GABA
release was blocked by 80 ± 13% (n = 4). In
parallel experiments, we measured intracellular [Ca2+] in
MIN6 m9 cells under similar conditions to above. Intracellular [Ca2+] in the resting cells amounted to 93 ± 3 nM (n = 62). L-Glutamate, AMPA,
and kainate at 500 µM each increased intracellular
[Ca2+], which corresponded to 109 ± 9 (n = 6), 117 ± 16 (n = 7), and 132 ± 18 nM (n = 15), respectively.
The kainate-evoked increase in intracellular [Ca2+] was
blocked upon incubation with EGTA-AM at 50 µM or with
nifedipine at 20 µM. Furthermore, replacement of
Na+ in the medium with
N-methyl-D-glucamine prevented the increase in
intracellular [Ca2+] by 88 ± 13%
(n = 20). Thus, the glutamatergic stimulation-evoked GABA secretion and increased intracellular [Ca2+] were
well correlated, indicating that stimulation of AMPA receptors triggers
Ca2+-dependent secretion of GABA under low
glucose conditions.
Glutamatergic stimulation-evoked GABA secretion was also observed with
the isolated islets (Fig. 6). The islets
were first incubated under 16.7 mM glucose conditions and
then transferred to 3.3 mM glucose conditions to facilitate
the secretion of L-glutamate from In this study, we presented evidence that We showed that both VGLUT1 and VGLUT2 are specifically localized with
glucagon-containing secretory granules in islet Several works have reported that L-glutamate or agonists of
glutamate receptor stimulate to some extent the secretion of insulin under high glucose conditions in cultured cells, isolated islets, and
perfused pancreas (4, 6, 7, 9). Consistently, we observed that
L-glutamate slightly stimulates (~10%) insulin secretion
from MIN6 m9 cells (Fig. 5B). However, the
L-glutamate-stimulated insulin secretion might not occur
under physiological conditions, since the high glucose conditions do
not trigger the L-glutamate signaling (Fig. 4), and
therefore, L-glutamate is not expected to become an
intercellular transmitter in islets under high glucose conditions.
Another significant finding obtained in the present study is that the
L-glutamate signaling triggers GABAergic response in clonal
The glutamatergic stimulation-evoked GABA secretion exhibits some
unique features as to the mode of L-glutamate signal
reception. At first, the glutamatergic signals become effective when
the glucose concentration in the medium decreases. This suggests that the ability of glutamate signal input of As to the physiological significance of the glutamatergic and GABAergic
signaling in the islets, we propose that these signaling pathways may
be involved in negative regulatory mechanism on glucagon secretion,
since the secreted GABA in turn binds to GABAA receptors on In conclusion, we solved critical issues, at least partly,
i.e. where, when, and how L-glutamate appears,
and what happen upon stimulation of glutamate receptors in the islets:
the low glucose conditions and To our knowledge, this is the first example of secretory
granule-mediated glutamatergic chemical transduction. Recently, we showed that D-aspartate is accumulated in secretory
granules and secreted from PC12 cells (48). Thus, co-secretion of
excitatory amino acids with hormones might be a common feature in
endocrine signal transmission.
cells (Hayashi, M., Otsuka, M., Morimoto, R.,
Hirota, S., Yatsushiro, S., Takeda, J., Yamamoto, A., and Moriyama, Y. (2001) J. Biol. Chem. 276, 43400-43406). Here we show
that VGLUT2 is specifically localized in glucagon-containing secretory
granules but not in synaptic-like microvesicles in
TC6 cells,
clonal
cells, and islet
cells. VGLUT1, another VGLUT isoform,
is also expressed and localized in secretory granules in
cells. Low
glucose conditions triggered co-secretion of stoichiometric amounts of
L-glutamate and glucagon from
TC6 cells and isolated
islets, which is dependent on temperature and Ca2+ and
inhibited by phentolamine. Similar co-secretion of
L-glutamate and glucagon from islets was observed upon
stimulation of
-adrenergic receptors with isoproterenol. Under low
glucose conditions, stimulation of glutamate receptors facilitates
secretion of
-aminobutyric acid from MIN6 m9, clonal
cells, and isolated islets. These results indicate that co-secretion of
L-glutamate and glucagon from
cells under low glucose
conditions triggers GABA secretion from
cells and defines the mode
of action of L-glutamate as a regulatory molecule for the
endocrine function. To our knowledge, this is the first example of
secretory granule-mediated glutamatergic signal transmission.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cells, glucagon-secreting
cells,
somatostatin-secreting
cells, and pancreatic polypeptide-secreting
F cells. These islet cells expresses functional glutamate receptors and
plasma membrane-type glutamate transporter (4-11), suggesting that
L-glutamate functions as an intercellular transmitter in
islet. In fact, L-glutamate has been shown to affect
secretion of insulin or glucagon from islet cells, isolated islets, or
perfused pancreas (4-11). However, the role of L-glutamate
as an intercellular chemical transmitter in the islets has been long
controversial, mainly because critical issues, i.e. where,
when, and how L-glutamate appears in the islets and what
happens upon stimulation of glutamate receptors in the islets, remain unresolved.
TC6 cells, clonal
cells, and islet
cells, but
not in
or
cells (18). These results are consistent with the
occurrence of Ca2+-dependent exocytosis of
L-glutamate from
TC6 cells (22) and suggest that
cells are the sites of L-glutamate signal appearance.
TC6 cells and islet
cells. Low glucose conditions triggers
co-secretion of stoichiometric amounts of L-glutamate and
glucagon. Stimulation of glutamate receptors in turn facilitates GABA
secretion from
cells. These results solve, at least in part, where,
when, and how L-glutamate appears in the islets and what
happens upon stimulation of glutamate receptors in the islets. Since
evidence for a role of GABA as a paracrine signal transmitter in the
islet has been reported (23-26), these results also suggest the
presence of L-glutamate- and GABA-mediated cross-talk
between
and
cells in the islets of Langerhans.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
TC6 cells were cultured as described (28). MIN6 m9 cells were cultured as
described (29).
TC6 cells (1.0 × 108 cells)
were washed with 20 mM MOPS-Tris (pH. 7.0) containing 0.3 M sucrose, 5 mM EDTA, 5 µg/ml leupeptin, and
5 µg/ml pepstatin A and then extensively homogenized. The homogenate
was centrifuged at 800 × g for 10 min, and the
resultant supernatant was centrifuged at 100,000 × g
for 30 min. The particulate fraction was suspended in the above buffer
and applied to a continuous sucrose density gradient (0.4-1.4
M) and centrifuged at 78,000 × g for
3.5 h. Then, the supernatant was fractionated in 11 tubes from the
bottom, and the glucagon content was determined (see Fig.
1C). Crude synaptic vesicles (LP2 fraction) were prepared as
described previously (18). To prepare membrane fraction of islets, at
least 500 islets were washed, suspended in 2 ml of 20 mM
MOPS-Tris (pH 7.0) containing 0.3 M sucrose, 5 mM EDTA, 10 µg/ml pepstatin A and 10 µg/ml leupeptin, and homogenized by hand with a small glass homogenizer. The homogenate was centrifuged at 900 × g for 10 min, and the
resultant supernatant was centrifuged at 266,000 × g
for 30 min in a Beckman Optima TLX ultracentrifuge. The pellet was
suspended in the same buffer and used for experiments.
20 °C. Ultra-thin sections on nickel grids were incubated with phosphate-buffered saline containing 2% goat
serum and 0.5% bovine serum albumin for 15 min and then treated with a
mixture of antibodies against VGLUT2 (50-diluted serum), glucagon
(1000-diluted), and insulin (200-diluted) or with a mixture of VGLUT2,
glucagon, and somatostatin (25-diluted) for 30 min. In the experiments
in Fig. 2D, a mixture of antibodies against VGLUT1
(50-diluted serum) and glucagon (1000-diluted) was used. Then the
sections were washed and treated with the secondary antibodies
conjugated with colloidal gold. Then the sections were washed with 0.1 M cacodylate buffer, postfixed with 5% glutaraldehyde, stained sequentially with uranyl acetate for 30 min and lead citrate for 1 min, and observed under a Hitachi H-7100S electron microscope.
-mercaptoethanol. Then Western
analysis was conducted as described (18).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
TC6 cells and islet
cells possess at least two
kinds of secretory vesicles, glucagon-containing secretory granules and synaptic-like microvesicles (SLMVs) (18). Indirect immunofluorescence microscopy indicated that VGLUT2 is co-localized with glucagon, a
marker of secretory granules, but not with synaptophysin, a marker of
synaptic vesicles or SLMVs, in
TC6 cells (Fig.
1, A and B).
Consistently, sucrose density gradient centrifugation of the
particulate fraction of
TC6 cells separated glucagon (fractions 3-7) and synaptophysin (fractions 7-11), indicating the separation of
glucagon-containing secretory granules and SLMVs (Fig. 1C). VGLUT2 is distributed similarly to glucagon but not to synaptophysin, which is in contrast with the co-localization of VGLUT2 and
synaptophysin in neuronal synaptic vesicles (Fig. 1C). In
the islets of Langerhans, triple labeling for immunoelectronmicroscopy
indicated that 15 nm of gold particles for VGLUT2 were specifically
associated with the membranes of secretory granules (Fig. 1,
D and E, and insets, arrowheads) that had been labeled with 5 nm of gold
particles for glucagon (Fig. 1, D and E, and
insets, arrows). In contrast, essentially no gold
particles for VGLUT2 were observed in any organelles, including
secretory granules in
or
cells in the islets (Fig. 1,
D and E, white arrows).
Quantitatively, the labeling densities for VGLUT2 in
glucagon-containing secretory granules, cytoplasm, and nucleus of
cells were 21.13 ± 1.99, 0.35 ± 0.12, and 0.85 ± 0.21 (number of immunogold particles/µm2, four independent
experiments), respectively. The labeling densities less than 0.79 ± 0.13 (number of immunogold particles/µm2, four
independent experiments) were also observed in secretory granules and
cytoplasm of
and
cells. Neither control serum nor antiserum
pre-absorbed with an antigenic peptide for VGLUT2 gave any specific
labeling (data not shown). Taken together, these results demonstrated
that VGLUT2 is associated with secretory granules in
TC6 cells and
islet
cells.
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Fig. 1.
VGLUT2 is present in
glucagon-containing secretory granules. A and
B, cultured TC6 cells were doubly immunostained with a
pair of antibodies against VGLUT2 and glucagon (A) or VGLUT2
and synaptophysin (B) and then observed under a confocal
laser microscope. Merged pictures are also shown. Bar, 10 µm in A, B. C, subcellular
fractionation revealed that VGLUT2 is associated with the secretory
granule fraction. Sucrose density gradient of ultracentrifugation of
the particulate fraction of
TC6 cells was conducted according to
"Experimental Procedures", and the glucagon content in each
fractions was determined. Aliquots (40 µl) were also subjected to
SDS-polyacrylamide gel electrophoresis, followed by Western blotting
with antibodies against VGLUT2 and synaptophysin (syn).
Crude synaptic vesicles (P2 fraction) were also fractionated as
described above, and immunoblotting of each fraction was performed.
D and E, triple gold labeling
immunoelectronmicroscopy of islets. D, VGLUT2 (15 nm in
diameter), insulin (10 nm in diameter), and glucagon (5 nm in
diameter). E, VGLUT2 (15 nm in diameter), somatostatin (10 nm in diameter), and glucagon (5 nm in diameter). VGLUT2 is associated
with secretory granules in
cells, while VGLUT2 is not present in
or
cells. Arrowheads and arrows indicate
labeling for VGLUT2 and glucagon, respectively. White arrows
indicate labeling for insulin in
cells and somatostatin in
cells. Insets in D and E, boxed areas
enlarged. Bar = 1 µm.
and
cells completely, we next examined the expression
and localization of VGLUT1, another VGLUT isoform specifically
expressed in neuron (14, 15).
TC6 cells is under detection limit in our
experimental systems (data not shown). However, unexpectedly, expression of the VGLUT1 gene in isolated islets was proven by RT-PCR
using a specific DNA probe (Fig.
2A). An amplified product with
the expected size and nucleotide and deduced amino acid sequences for
VGLUT1 was obtained. Western blotting with specific antibodies for
VGLUT1 detected a single islet polypeptide exhibiting similar migration
to that of neuronal VGLUT1 (Fig. 2B) (14, 15).
Immunohistochemistry on the frozen sections of islets revealed that
VGLUT1 is co-localized with glucagon but not insulin or somatostatin
(Fig. 2C). Together, these results indicate that VGLUT1 is
also expressed in
cells but not in
or
cells. Expression of
VGLUT1 was somewhat heterogeneous among
cells, and some
cells
showed intense immunoreactivity in contrast with ubiquitously intense
expression of VGLUT2 (18).
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Fig. 2.
Expression and localization of VGLUT1
in islets. A, expression of the VGLUT1 gene in brain (lanes
1, 3) and isolated islets (lanes 2,
4) was detected by RT-PCR. No amplified products were
obtained without the RT reaction (lanes 3 and 4).
B, Western blotting indicates the presence of VGLUT1 in
islets. Lanes 1 and 3, crude synaptic vesicles
from rat brain (20 µg); lanes 2 and 4, islets
(30 µg each). In lanes 3 and 4, antibodies
preabsorbed with antigenic peptides (2 mg) were used. C,
sections of pancreas were doubly immunostained with antibodies against
VGLUT1 and glucagon, VGLUT1 and insulin, or VGLUT1 and somatostatin,
and then observed under a confocal laser microscope. Merged pictures
are also shown. Bar = 20 µm. D, double
gold labeling immunoelectronmicroscopy of islets. Arrowheads
and arrows indicate labeling for VGLUT1 (15 nm in diameter)
and glucagon (5 nm in diameter), respectively. Bar = 500 nm.
or
cells in the
islets (data not shown). Taken together, it is concluded that both
VGLUT1 and VGLUT2 are expressed in islet
cells and only VGLUT2 is
expressed in
TC6 cells and that both VGLUTs are specifically
localized with glucagon-containing secretory granules.
cells under low glucose conditions.
Consistently, the L-glutamate immunoreactivity coincided
with that of VGLUT2 in the islets (Fig.
3). The L-glutamate
immunoreactivity decreased to the background level when the antibodies
preabsorbed with L-glutamate conjugated with bovine serum
albumin were used (Fig. 3D).
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Fig. 3.
Immunological co-localization of
L-glutamate and VGLUT2 in the islet. An islet was
immunostained for L-glutamate (A) and VGLUT2
(B), and then observed under a confocal laser microscope.
Merged picture is also shown as C. In D,
antibodies against L-glutamate after incubation with 1 mg/ml bovine serum albumin-L-glutamate for overnight were
used. Bar = 50 µm.
TC6 cells were incubated with 16.7 mM glucose and then transferred to low glucose conditions,
i.e. 3.3 mM. It was found that appreciable
amounts of L-glutamate as well as glucagon were secreted
from the islets: 54.3 ± 2.9 pmol L-glutamate per
islet at 30 min, corresponding to about 53% of total
L-glutamate, was released (Fig.
4A, open circles).
When the islets were transferred to the same glucose conditions at 16.7 mM, about 30% L-glutamate release was observed
at 30 min (Fig. 4A, closed circles). Essentially the same basal level of L-glutamate release was observed
when islets were first incubated with 3.3 mM glucose and
then transferred to glucose solution at 16.7 mM (data not
shown). Thus, L-glutamate release from the islets
constitutes the low glucose-stimulated and glucose-independent one. The
low glucose-stimulated L-glutamate release disappeared at
below 20 °C or in the presence of EGTA, indicating that the low
glucose-stimulated L-glutamate release was dependent on
Ca2+ and temperature (Fig. 4A, open
squares). Phentolamine, which inhibits the exocytosis of glucagon
via Gi2-dependent activation of
calcineurin (34), inhibited the low glucose-stimulated and Ca2+-dependent L-glutamate
secretion in a parallel manner (Fig. 4, open triangles). The
ratio of the low glucose-stimulated and
Ca2+-dependent secretion of
L-glutamate and glucagon was always stoichiometric, being
1229 ± 105 (mol/mol, n = 4). Together, these
results strongly suggest that the low glucose-stimulated
L-glutamate release is due to exocytosis of
glucagon-containing secretory granules. In contrast, the
glucose-independent L-glutamate release was
Ca2+-independent, suggesting that it is due to nonspecific
leakage from islet cells.
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Fig. 4.
Low-glucose conditions trigger co-secretion
of L-glutamate and glucagon from islets. Islets (20 pieces per assay) were incubated in a Ringer's solution containing
16.7 mM glucose for 1 h. Then, the glucose
concentration was changed to 3.3 mM (all open
symbols) or 16.7 mM (closed circles). In
open triangles), islets (20 pieces per assay) were incubated
in a Ringer's solution containing 16.7 mM glucose for
1 h. Then, the glucose concentration was changed to 3.3 mM in the presence of 100 µM phentolamine
according to Ref. 34. In open squares, islets (20 pieces per
assay) were incubated in a Ringer's solution containing 16.7 mM glucose for 1 h. Then, the glucose concentration
was changed to 3.3 mM in the presence of EGTA.
Ca2+ in the medium was reduced to 40 nM. Then
the medium was sampled at the times indicated, and the concentrations
of L-glutamate (A) and glucagon (B)
released from islets were measured. All the results are means ± S.E. (four independent experiments).
TC6 cells: 2.6 ± 0.3 nmol L-glutamate and 6.8 ± 0.7 ng glucagon per 106 cells per hour
(n = 6) were secreted. In this case, the
L-glutamate released corresponded to about 23% of total
L-glutamate, the stoichiometry being 1345 ± 208.
-adrenergic receptors on
cells irrespective
of glucose conditions (35). As expected, even when the islets were
incubated with 16.7 mM glucose condition, isoproterenol
triggered the stoichiometric secretion of L-glutamate
(35.9 ± 2.3 pmol/islet, n = 4) and glucagon (0.091 ± 0.005 ng/islet, n = 4) at 30 min. The
isoproterenol-evoked release of L-glutamate and glucagon
was observed irrespective of glucose conditions employed, totally
Ca2+-dependent, and inhibited by 91 ± 3 and 95 ± 3% (n = 4), respectively, by
propranolol, a
blocker, at 10 µM. Thus, either the
low glucose conditions or stimulation of
-adrenergic receptors
triggers release of stoichiometric amounts of L-glutamate
and glucagon from isolated islets and
TC6 cells.
Cells
under Low Glucose Conditions--
What happens upon stimulation of
glutamate receptors in the islets?
cells may receive the glutamate
signal since they express glutamate receptors (6-8, 10, 11). It has
been shown that
cells store GABA in SLMVs, but not insulin granules
(36, 37), and secrete it through a
Ca2+-dependent exocytotic pathway (32, 33). We
investigated whether or not L-glutamate affects GABA
secretion from
cells.
cells that retain
glucose-responsive insulin secretion capacity (29). Consistently, the cells could secrete insulin (205 ± 8 ng/106 cells at
30 min, four independent experiments) when they were first incubated
with 3.3 mM glucose and then transferred to 16.7 mM glucose (Fig. 5). Under
other glucose conditions, e.g. the cells were first
incubated with 16.7 mM glucose and then transferred to 16.7 mM or 3.3 mM glucose or the cells were
incubated with 3.3 mM glucose throughout, a decreased level
or only a background level of insulin secretion was observed (Fig. 5).
MIN6 m9 cells also secrete GABA: on average, 0.62 ± 0.07 nmol of
GABA/106 cells at 30 min, which corresponds to about 6% of
total GABA, was secreted when the cells were first incubated with 16.7 mM glucose and then transferred to 16.7 mM or
3.3 mM glucose, or the cells were incubated with 3.3 mM glucose throughout (Fig. 5). GABA secretion was
stimulated about 1.2-fold, when they were first incubated with 3.3 mM glucose and then transferred to 16.7 mM
glucose (Fig. 5). Thus, GABA secretion is not significantly dependent
on the glucose conditions as compared with insulin secretion, confirming the presence of distinct secretory pathways for insulin and
GABA, as shown by the published works (31-33).
View larger version (18K):
[in a new window]
Fig. 5.
Secretion of GABA and insulin from MIN6 m9
cells. Secretion of GABA (A) and insulin (B)
under different glucose conditions was examined in the presence or
absence of L-glutamate or an agonist or antagonist of
glutamate receptors. The cells were first incubated with 16.7 or 3.3 mM sucrose and then transferred to 16.7 or 3.3 mM glucose, as specified, in the presence of
L-glutamate, AMPA, or kainate at 0.5 mM. After
30 min, GABA and insulin in the medium were determined. In some
experiments, CNQX at 50 µM was also included. 100% GABA
corresponds to 0.58 ± 0.05 nmol/106 cells
(n = 4). 100% insulin release corresponds to 14.4 ± 0.6 ng/106 cells (n = 4). An
asterisk indicates that a value is significantly different
from the value obtained in a control experiment (none) without an
agonist or antagonist under each set of incubation conditions with
Student's t test (*, p < 0.05; **,
p < 0.01).
cells as described in
the previous section. About 2-fold GABA was released from the islets as
compared with the control level, i.e. the value obtained for
islets with 16.7 mM glucose. The low glucose-responsive
GABA release was sensitive to CNQX, suggesting that endogenous
L-glutamate from
cells triggers GABA secretion.
Exogenous L-glutamate and kainate stimulated the GABA release about 1.1-1.5-fold as compared with the absence of these compounds. CNQX decreased the L-glutamate- and
kainate-evoked GABA release to the control levels. Consistent with the
results for MIN6 m9 cells, under high glucose conditions, neither GABA secretion nor insulin secretion was stimulated by the addition of
either L-glutamate or kainate.
View larger version (17K):
[in a new window]
Fig. 6.
Secretion of GABA and insulin from isolated
islets. Isolated islets (20 pieces per assay) were incubated as
described in the legend to Fig. 5, and then GABA (A) and
insulin (B) release were quantified. 100% GABA corresponds
to 1.3 ± 0.1 nmol/106 cells (n = 4).
100% insulin release corresponds to 15.1 ± 0.6 ng/106 cells (n = 4). An
asterisk indicates that a value is significantly different
from the value obtained in a control experiment (none) without an
agonist or antagonist under each set of incubation conditions with
Student's t test (*, p < 0.05; **,
p < 0.01).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cells are
glutamatergic in nature and indicated the conditions when and how
L-glutamate appears. L-Glutamate is co-stored
with glucagon in secretory granules in
cells. Then, the low glucose
conditions facilitate secretion of L-glutamate and glucagon
so as to trigger the glutamatergic signaling in the islet. In our
previous study, we proposed that SLMVs in
TC6 cells are responsible
for storage and secretion of L-glutamate (22). However,
combined immunohistochemistry and immunoelectronmicroscopy clearly
indicate that glucagon-containing secretory granules, but not SLMVs,
are the sites for L-glutamate storage in
TC6 cells and
islet
cells. These results are consistent with observation by Tong
et al., although they did not identify the VGLUT2-positive
vesicles as glucagon-containing secretory granules in
cell
(11).
cells. L-Glutamate may be accumulated in glucagon-containing
secretory granules by active transport through VGLUTs at the expense of an electrochemical gradient of protons across the membrane, which is
established by vacuolar H+-ATPase. Since apparent
difference in transport properties between VGLUT1 and VGLUT2 has not
been obtained yet (14-21), the VGLUT isoforms in
cells may be
simply responsible for the storage of L-glutamate.
Localization of VGLUTs with glucagon-containing secretory granules
predicts the mode of L-glutamate signal output. In fact, we
next showed that L-glutamate immunoreactivity is
co-localized with VGLUT2 (Fig. 3). Moreover, we showed that either low
glucose conditions or stimulation of
receptors on
cells
actually triggers release of stoichiometric amount of
L-glutamate and glucagon with similar Ca2+ and
temperature dependence and drug sensitivities. Overall, we concluded
that L-glutamate is co-stored and co-secreted with glucagon from
cells under low glucose conditions.
cells should be
regarded as L-glutamate-secreting endocrine cells.
cells and isolated islets. The released L-glutamate may bind to the corresponding receptors on the islet cells, causing a
paracrine or autocrine response (3-11). Islet
cells contain GABA
in SLMVs at concentrations comparable level to the central nervous
systems (36, 37). Upon depolarization,
cell secretes GABA through
Ca2+-dependent exocytosis (32, 33), and the
released GABA becomes a paracrine or autocrine transmitter (24-26).
However, the mode of action of GABA as an intercellular chemical
transmitter, especially the timing for its appearance with the
receptor-accessible manner in islet, was less understood. Previous
studies on GABA release have been performed after a long incubation
period, around a day (31, 38, 39). Under such conditions, amounts of
GABA release may reflect the metabolic state of
cells but not
directly reflect the rate of GABA secretion through exocytosis of
SLMVs. In contrast, depolarization-evoked exocytosis of GABA-containing
SLMVs seems to be completed within around 10-20 min (32, 33). We found that L-glutamate, AMPA or kainate stimulates GABA release
from clonal
cells and isolated islets. The properties of the GABA secretion, e.g. time course, temperature, and
Ca2+-dependences and sensitivities to Ca2+
channel blockers are similar to those of GABA secretion through depolarization-evoked exocytosis of SLMVs (32, 33). Thus, it is
concluded that the L-glutamate triggers GABA secretion
through enhanced exocytosis of GABA-containing SLMVs. It is noteworthy that our results indicate for the first time that
cells and
cells communicate together by way of L-glutamate- and
GABA-mediated signaling. The glutamatergic signaling and resultant
GABAergic signaling cease when the blood glucose concentration
increases because of voltage-dependent inhibition of
Na+ channel due to closure of the K+-ATP
channel of
cells (40). Then, the exocytosis of insulin is
facilitated through KATP channel-mediated depolarization of
cells
(40).
cells changes with glucose
concentration, and then
cells can receive L-glutamate signals only when
cells secrete L-glutamate. Although
we can not explain the molecular mechanism underlying the
glucose-dependent change on L-glutamate signal
reception at present, one plausible explanation is that AMPA receptors
on
cells take on agonist-accessive and -inaccessive forms depending
on the glucose concentrations. Another important feature is that the
glutamatergic stimulation selectively triggers GABA secretion and does
not facilitate exocytosis of insulin granules. Although the exocytosis
of GABA-containing SLMVs and insulin granules requires an increase in
intracellular [Ca2+] (this study and Refs. 32 and 41),
the present results strongly suggest that the exocytosis of these two
kinds of secretory vesicles is differently regulated. Recently,
synaptotagmins were reported to form a hierarchy of exocytotic
Ca2+ sensors with distinct Ca2+ affinities
(42). Furthermore,
cells express various kinds of synaptotagmin
isoforms involved or not involved in insulin exocytosis (43-45). It is
possible that synaptotagmins and/or the related proteins associated
with SLMVs may cause a secretory response distinct to that of insulin
granules. Both possibilities are now under investigation in our laboratory.
cells,
causing inhibition of glucagon secretion (25, 26, 46). Consistent with
the idea, stimulation of metabotropic glutamate receptor type 8 (mGluR8), a class III receptor, on
cells, strongly inhibited
glucagon secretion under the low glucose condition (11). In this case,
L-glutamate may function as an autocrine-type chemical transmitter. We predict that L-glutamate also triggers
somatostatin secretion since
cells express AMPA type receptors (8),
and somatostatin inhibits glucagon secretion by way of the somatostatin receptors on
cells (47). We are now investigating this possibility in more details.
adrenergic stimulation trigger
L-glutamate secretion, and the released
L-glutamate in turn triggers GABA secretion in the isolated
islets. We presented the direct evidence that
cells and
cells
mutually interact by way of L-glutamate- and
GABA-signaling. Although the results obtained in vitro assay conditions may not necessary apply to native islet of Langerhans, it is
probable that in vivo changes of blood glucose concentration directly regulate the glutamatergic signal transmission in the islets.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. S. Takamori and Prof. R. Jahn
(Max-Plank Institute for Biophysical Chemistry) for providing an
expression plasmid for VGLUT1, Prof. S. Seino (Chiba University) and
Dr. K. Hamaguchi (Oita University) for their kind supply for MIN6 m9
cells and TC6 cells, respectively.
![]() |
FOOTNOTES |
---|
* This study was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan, Core Research for Evolutional Science, the Yamanouchi Foundation for Research on Metabolic Disorders, the Takeda Science Foundation, and the Umami Research Foundation.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.
§ Supported by a Research Fellowship from the Japan Society for the Promotion of Science for Young Scientists.
¶ Present address: Dept. of Neuroscience, Graduate School of Medicine and Dentistry, Okayama University, Okayama 700-8558, Japan.
Supported by a Research Fellowship from the Japan Society for
the Promotion of Science for Young Scientists.
§§ To whom correspondence should be addressed. Tel.: 81-86-251-7933; Fax: 81-86-251-7933; Email: moriyama@pheasant.pharm.okayama-u.ac.jp.
Published, JBC Papers in Press, October 31, 2002, DOI 10.1074/jbc.M206758200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
VGLUT, vesicular glutamate transporter;
MOPS, 4-morpholinepropanesulfonic
acid;
RT, reverse transcription;
DMEM, Dulbecco's modified Eagle's
medium;
GABA, -aminobutyrate;
AMPA, (RS)-
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid;
CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione;
SLMV, synaptic-like
microvesicles.
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