Departments of 1 Pediatrics and 2 Physiology, Steele Memorial Children's Research Center, University of Arizona Health Sciences Center, Tucson, Arizona 85724
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ABSTRACT |
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Glutamate has been
suggested to play an important role in the release of insulin and
glucagon from pancreatic cells via exocytosis. Vesicular glutamate
transporter is a rate-limiting step for glutamate release and is
involved in the glutamate-evoked exocytosis. Two vesicular glutamate
transporters (VGLUT1 and -2) have recently been cloned from the brain.
In this report, we first functionally characterized vesicular glutamate
transporter in cultured pancreatic - and
-cells, and then
detected mRNA expression of VGLUT1 and -2 in these cells. We also
investigated the effect of high or low level of glucose on vesicular
glutamate transport in cultured pancreas cells. Our results suggest
that both
- and
-cells contain functional vesicular glutamate
transporter. The transport characteristics are similar to the cloned
neuronal VGLUT1 and -2 in regard to ATP dependence, substrate
specificity, kinetics, and chloride dependence. VGLUT2 mRNA is
expressed in both
- and
-cells, whereas VGLUT1 is only expressed
in
-cells. High (12.8 mM) and low (2.8 mM) concentrations of glucose
increased vesicular glutamate transport in
- and
-cells,
respectively. VGLUT2 mRNA was significantly increased in
- and
-cells by high and low glucose concentration, respectively.
This increase in VGLUT2 mRNA was suppressed by actinomycin D. We
conclude that both
- and
-cells possess functional vesicular glutamate transporters regulated by alteration in glucose
concentration, partly via the transcriptional mechanism.
diabetes; insulin; glucagon; transcriptional regulation
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INTRODUCTION |
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L-GLUTAMATE IS THE MAJOR
EXCITATORY neurotransmitter in the mammalian central nervous
system and plays important roles in many neuronal processes, such as
fast synaptic transmission and neuronal plasticity. More recently, it
has been suggested that glutamate is a functional molecule in
nonneuronal tissues including pancreas, bone, stomach, intestine,
liver, lung, kidney, and skin (28). Glutamate has been
found to stimulate insulin (5, 7) and glucagon (6,
17) secretion in the pancreatic - and
-cells, respectively. In the model of glucose-induced insulin secretion, increased cytosolic Ca2+ concentration by the opening of
voltage-sensitive Ca2+ channels, constitutes the main
trigger of insulin exocytosis. However, the Ca2+ signal
alone is not sufficient for the full development of biphasic insulin
secretion (see Ref. 20 for review). Maechler and Wollheim (19) recently provided evidence that glutamate acts
downstream of the mitochondria by sensitizing the
Ca2+-mediated exocytotic process. In that model, a rise in
extracellular glucose causes elevation of intracellular glucose
followed by a subsequent increase in glycolysis and tricarboxylic acid
activity. These metabolic changes lead to an increase in cellular ATP
that closes ATP-sensitive potassium channels causing depolarization of
the plasma membrane potential. Subsequently, depolarization opens
voltage-sensitive Ca2+ channels, raising intracellular
Ca2+ concentration and triggering insulin exocytosis.
Glutamate is packaged in vesicles with insulin by the pancreatic cells,
and the glutamate sensitizes the release of insulin.
Neuronal cells utilize L-glutamate as an intracellular
signaling molecule via glutamatergic systems comprising the storage of
glutamate in synaptic vesicles and its exocytosis, glutamate receptor,
and glutamate reuptake mechanism. It has been suggested that Langerhans
islets have their own glutamatergic system as do neurons. Multiple
glutamate receptors have been found in the pancreas (14, 15, 18,
33) and glutamate has been found to stimulate insulin and
glucagon release via -amino-3-hydroxy-5-methylisoxazole-4-propionic acid subtypes of the glutamate receptor (5, 6). A
sodium-dependent glutamate transporter has been identified as the
glutamate reuptake system on the plasma membrane of the pancreas
(34). However, vesicular glutamate transporter, the
protein responsible for the accumulation of glutamate from cytoplasm
into the vesicles, has not been characterized in the pancreas.
Glutamate uptake into the secretory granules by vesicular glutamate
transporter is a rate-limiting step for glutamate release. Vesicular
glutamate transporter has been characterized in neurons and
pinealocytes, which possess an active glutamate-specific transporter dependent on ATP-generated electrochemical proton gradient across the
vesicle membrane, on extravesicular Cl concentration, and
on temperature (13, 21-23). Vesicular glutamate transport processes depend on the proton electrochemical gradient (
µH+) generated by the Mg2+-activated
vacuolar H+-ATPase (V-ATPase) on the vesicular membrane
(12). When protons are pumped into the vesicular lumen, a
proton gradient (
pH) and a membrane potential (
) occur across
the membrane to form
µH+, which favors the exchange of
luminal protons for the cytoplasmic transmitter (8, 21,
23). Two vesicular glutamate transporters, VGLUT1 and -2, with
75% homology, have been recently identified from synaptic vesicles of
neurons (3, 4, 30). Both transporters have functional
characteristics of the synaptic vesicle glutamate transporter,
including ATP dependence, chloride stimulation, substrate specificity,
and substrate affinity. These molecular and functional advances led us
to functionally characterize and identify vesicular glutamate
transporter(s) from the pancreatic
- and
-cells and to study the
regulation of these transporters in response to the changes of glucose concentration.
This is the first study to functionally characterize the vesicular
glutamate transporter in the pancreatic cells. We found that both -
and
-cells contained functional vesicular glutamate transporters
similar to the cloned VGLUT1 and -2 regarding ATP dependence, substrate
specificity, kinetics, and chloride dependence. VGLUT2 is expressed in
both
- and
-cells and is regulated by alteration of extracellular
glucose concentration.
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MATERIAL AND METHODS |
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Materials. N,N'-dicyclohexylcarbodiimide (DCCD) was purchased from Fisher Scientific (Pittsburgh, PA). All other chemicals were obtained from Sigma (St. Louis, MO).
Cell culture, preparation of vesicular membranes, and glutamate
uptake.
Two -cell lines (mouse
-TC-1-9 and human HPAC), two
-cell
lines (mouse
-TC-6 and rat RIN-m), and a rat pheochromocytoma cell
line (PC-12) were purchased from American Type Culture Collection (Manassas, VA). Cells were maintained in the manufacturer's
suggested culture medium (Ham's F-12K medium for PC-12 cells, a 1:1
mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium
for HPAC cells, Dulbecco's modified Eagle's medium for
-TC-1-9 and
-TC-6 cells, and RPMI 1640 medium for RIN-m
cells). Various glucose concentrations will be indicated in the figure
legends for the individual experiment. For experiments dealing
with glucose regulation,
-TC-1-9 and
-TC-6 cells were
cultured in Dulbecco's modified Eagle's medium supplemented with 7.5 mM glucose, 10% fetal bovine serum, and 1% penicillin and
streptomycin. At 70% confluence, the cells were incubated in
Dulbecco's modified Eagle's medium containing either 12.8 (
-TC-6
cells) or 2.8 mM (
-TC-1-9 cells) glucose for 12 h before
they were harvested. For transcriptional assays, the cells were
pretreated with actinomycin D (5 µg/ml) (Calbiochem-Novabiochem, San
Diego, CA) for 2 h and then were treated with 12.8 or 2.8 mM
glucose medium in the presence of actinomycin D before they were harvested.
mRNA isolation and semiquantitative RT-PCR.
mRNAs were isolated from four types of cells incubated at different
glucose concentrations by using the Fast-Track mRNA purification kit
(Invitrogen, Carlsbad, CA). First-strand cDNAs were synthesized by
using oligo(dT) primer. Subsaturated levels of cDNA templates that were
needed to produce a dose-dependent amount of PCR products were defined
in the initial experiments by testing a range of template
concentrations. Subsequent PCR was carried out with subsaturated level
of RT reaction with specific primers for VGLUT1 and -2 of mouse, human,
and rat species and -actin in separate reactions for 35 cycles
(94°C for 1 min, 55°C for 1 min, and 72°C for 1 min). The primers
were designed to amplify a 453-bp region corresponding to amino acids
368-519 for human VGLUT1 (GenBank accession no. AB032436) and
an 870-bp region corresponding to amino acids 290-580
for VGLUT2 (GenBank accession no. AF 324864). The sequences of
primers are mouse VGLUT1: sense strand
5'-CACATAATGTCCACTACCAA-3', antisense strand
5'-CACTGCCAGCCAGCTGGTCG-3'; human VGLUT1: sense strand
5'-CGCATCATGTCCACCACCAA-3', antisense strand
5'-CACTGCCAGCCAGCTGGTCA-3'; mouse VGLUT2: sense strand
5'-ATCTGCTAGGTGCAATGGAA-3', antisense strand
5'-AATCATCTCGGTCCTTATAG-3'; and human VGLUT2: sense strand 5'-ATCTTTTAGGTGCAATGGAA-3', antisense strand
5'-AATCAACTCGGTCCTTATAG-3'. We used mouse primers to amplify rat VGLUT1
and -2 message, because the sequences of the primers are identical
between these two spices. PCR products from the same cell line were
loaded on 1% agarose gels and visualized with ethidium bromide. PCR
products were sequenced at the University of Arizona sequencing
facility to ensure the correct sequences.
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RESULTS |
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Functional characterization of vesicular glutamate transport in
cultured pancreatic - and
-cell lines.
Vesicular glutamate transport has been extensively characterized in the
neuronal synaptic vesicles. The neuronal vesicular ATP-dependent
glutamate transporter is specific for glutamate, is stimulated by
millimolar concentrations of chloride, and has a low affinity for
uptake (Km, ~1-2 mM) (16,
24). We first tested two
-cell lines (mouse
-TC-1-9
and human HPAC) and two
-cell lines (mouse
-TC-6 and rat RIN-m)
for vesicular glutamate uptake. Figure
1A shows that vesicle
membranes from these four cell lines exhibited four- to fivefold
increased uptake of [3H]glutamate than that of PC-12
cell, a vesicular glutamate transporter null cell (3). We
then further characterized vesicular glutamate transport in
-TC-1-9 and
-TC-6 cells.
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mRNA expression of VGLUT1 and -2 in cultured pancreatic - and
-cells.
VGLUT1 and -2 were originally cloned from the brain and VGLUT2 protein
was shown to be expressed in the pancreatic islets (14).
However, the mRNA expression of these two transporters in
individual cells of islets has not been determined. Because functional vesicular glutamate transport was detected in the two
-cell lines (mouse
-TC-1-9 and human HPAC) and two
-cell
lines (mouse
-TC-6 and rat RIN-m) (Fig. 1A), we measured
endogenous mRNA expression of VGLUT1 and -2 in these cells by RT-PCR by
using human-, mouse-, or rat-specific primers corresponding to
different cell types. RT-PCR was performed with a subsaturated dose of
mRNA and PCR cycle, as determined by measuring
-actin mRNA (data not shown) (2). VGLUT2 was expressed in all types of tested
cells, although relatively less expression was observed in the two
-cell lines (Fig. 3, lanes
1-4). In contrast,
VGLUT1 was only expressed in the two
-cell lines (Fig. 3,
lanes 3 and 4). All PCR products were confirmed
by automatic sequencing. Neither VGLUT1 nor -2 was detected in RT
reaction-free negative control (data not shown). These results
suggested that VGLUT2 could play a functional role in the regulation of
vesicular glutamate uptake in both
- and
-cells, whereas VGLUT1
might play a role in a
-cells but not in an
-cells.
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Regulation of vesicular glutamate transport by changing of
extracellular glucose concentration.
Under normal physiological conditions, changes of serum glucose
concentration, i.e., hyperglycemia or hypoglycemia, result in secretion
of insulin or glucogan from the pancreas, respectively. Vesicular
glutamate transporter in pancreatic islet cells controls accumulation
and storage of glutamate in secretory vesicles. Therefore, we studied
the regulation of pancreatic vesicular glutamate transporter by
different glucose concentrations. Time courses of vesicular glutamate
transport in -TC-1-9 and
-TC-6 cells in response to high
(12.8 mM) and low (2.8 mM) glucose concentrations were shown in Fig.
4. Cells were maintained at physiological
glucose concentration (7.5 mM) before changing glucose concentration.
Under low glucose concentration (Fig. 4A), vesicular
glutamate uptake by
-TC-1-9 cells was significantly increased
after 12 h exposure and remained steady until 48 h. However,
vesicular glutamate uptake by
-TC-6 cells was not changed by the
same exposure. Interestingly, under high glucose concentration (Fig.
4B), vesicular glutamate uptake by
-TC-6 cells was
significantly increased after 12 h exposure, whereas vesicular
glutamate uptake by
-TC-1-9 cells was not changed by the same
exposure.
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Potential mechanism of glucose-mediated regulation of vesicular
glutamate transport in cultured pancreatic - and
-cells.
Because increased vesicular glutamate transport was observed after 12 h
exposure to low or high glucose concentrations, we tested the potential
mechanism for this regulation. As we have shown in Fig. 3,
-TC-1-9 cells expressed VGLUT2 but not VGLUT1. We first
determined VGLUT2 mRNA change in
-TC-1-9 cells in response to
low glucose level by the semiquantitative RT-PCR technique (Fig.
5). The expression of VGLUT2 mRNA was
significantly increased in
-TC-1-9 cells by low glucose (2.8 mM) stimulation, and the increased expression was suppressed by
pretreatment of actinomycin D.
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DISCUSSION |
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Glutamate plays an important role in glucose-induced insulin
secretion in pancreatic -cells and glucagon secretion in pancreatic
-cells by sensitizing Ca2+-induced hormone exocytosis.
Packaging and storage of glutamate into specialized secretory vesicles
in neurons and endocrine cells ensure its regulated release. A unique
group of proteins found on secretory vesicles is the transporter needed
for the accumulation of glutamate from cytoplasm into these vesicles.
Although Langerhans islets were believed to equip their own
glutamatergic systems, the functional characteristics of vesicular
glutamate transport in the pancreas has not been characterized. In this
report, we functionally characterized vesicular glutamate transporters
in pancreatic
- and
-cells, which appear to have the same major characteristics as their neuronal counterpart. VGLUT1 is expressed in
-cells, whereas VGLUT2 is expressed in both
- and
-cells. VGLUT2 is upregulated by changes of glucose concentration in both cells. We conclude that both
- and
-cells contained functional vesicular glutamate transporter(s), which can be regulated by alteration of glucose concentration.
Vesicular glutamate transport has been extensively characterized in
neuronal synaptic vesicles. This vesicular transport processes depend
on the µH+ generated by a Mg2+-activated
V-ATPase on the vesicular membrane (12). The vesicular ATP-dependent glutamate transporter is specific for glutamate, is
stimulated by millimolar concentrations of chloride, and has a low
affinity for uptake (Km
1-2 mM)
(16, 24). Endocrine cells, such as pancreatic
- and
-cells, contain synaptophysin-containing vesicles in which classical
neurotransmitters are accumulated (31). These vesicles
contain transporters for specific neurotransmitters and the transport
relies on the presence of ATP. Consistent with this, vesicular
glutamate transport in cultured pancreatic cells is specific for
glutamate and is ATP dependent. Bafalomycin A1 and DCCD, inhibitors for
vacuolar Mg2+-ATPase, dramatically inhibited glutamate
uptake. The plasma membrane and mitochondrial ATPase inhibitors,
oligomycin and ouabain, had no effect on glutamate uptake. Furthermore,
pancreatic vesicular glutamate transport is saturable with a
Km of 1.4 and 1.6 mM for
-TC-1-9 cells
and
-TC-6 cells, respectively.
The requirement of low chloride concentration is a significant property
of vesicular glutamate transport. Presence of chloride at low
concentrations (1-5 mM) is essential for the uptake of glutamate,
with substantially lower transporter activity observed at higher and
lower levels (10, 21, 29). This biphasic effect of
chloride has been observed in glutamate uptake from both
-TC-1-9 and
-TC-6 cells. The intracellular chloride
concentration is within 2-15 mM under normal physiological
conditions (1, 27). Thus the low extravesicular
concentration of chloride favoring vesicular glutamate uptake is
considered physiologically relevant in the cells. We did not test the
concentration of chloride in the pancreatic cells. However, the
chloride concentration in these cells is likely comparable to that of
neurons, because these endocrine cells have the major characteristics
of neurons, such as neurotransmitter systems. These results suggested
that the pancreatic vesicular glutamate transporter possessed similar
characteristics to their neuronal counterpart including ATP dependence,
transport kinetics, substrate specificity, and chloride dependence.
Because transport characteristics of the pancreatic vesicular glutamate
transporter are also similar to the two recently cloned neuronal
vesicular glutamate transporters, VGLUT1 and -2, expression of VGLUT1
and -2 was determined in the cultured pancreatic cells. VGLUT1 was
expressed only in -cells, whereas VGLUT2 was expressed in both
-
and
-cells, although a lesser level expression was found in
-cells. Similar to our study, another group (14) using double immunostaining with antibodies against VGLUT2 and glucagon found
that VGLUT2 protein is expressed in the pancreatic
-cell. However,
they failed to detect VGLUT2 protein in
-cells by using double
staining with antibody against VGLUT2 and insulin. This difference is
probably due to lesser sensitivity of immunohistochemistry for
detection of a protein with lower expression in the cells. The
differential expression of VGLUT1 and -2 implies that VGLUT2 may be the
vesicular glutamate transporter involved in regulation of glucagon
secretion from
-cells, because VGLUT1 is not present in
-cells.
Considerable evidence indicates that the biosynthesis and transport of
neurotransmitters undergo regulation by physiological or
pathophysiological factors. For example, vesicular monoamine transporter isoform 2 (VMAT2) is an important vesicular transporter for histamine. Recent work has demonstrated that VMAT2 gene
expression may be regulated by gastrin (32) and ovarian
hormones (26). Intracellular calcium increases vesicular
monoamine transporter through a transcriptional activation mechanism in
chromaffin cells (9). We have shown that VGLUT2 mRNA is
upregulated by high concentration of glucose in -cells and by low
concentration of glucose in
-cells. In contrast, expression of
VGLUT1 was not changed by high glucose, although its expression was
more predominant than VGLUT2 in
-cells. These findings suggest that
chronic exposure to low glucose concentration stimulates glutamate
uptake into secretory vesicles in
-cells, which favors
glutamate-evoked glucagon release, whereas chronic exposure to high
glucose concentration stimulates glutamate uptake into secretory
vesicles in
-cells, which favors glutamate-evoked insulin release.
However, the exact mechanisms of this regulatory processes are not
clear. The suppression of glucose-induced upregulation of VGLUT2 by
actinomycin D suggested that transcriptional mechanism was at least
partially involved. Further studies of regulation of VGLUT2 promoter by
glucose will help to elucidate the mechanism.
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ACKNOWLEDGEMENTS |
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This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant 2R01-R37-DK-33209.
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FOOTNOTES |
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Address for reprint requests and other correspondence: F. K. Ghishan, Professor and Head, Dept. of Pediatrics, Director, Steele Memorial Children's Research Center, 1501 N. Campbell Ave., Tucson, AZ 85724 (E-mail: fghishan{at}peds.arizona.edu).
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.
First published November 20, 2002;10.1152/ajpgi.00333.2002
Received 8 August 2002; accepted in final form 18 November 2002.
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