Diabetes Research Center, Vrije Universiteit Brussel, B-1090 Brussels, Belgium
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ABSTRACT |
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Pancreatic -cells express glutamate
decarboxylase (GAD), which is responsible for the production and
release of
-aminobutyric acid (GABA). Over a 24-h culture period,
total GABA release by purified rat
-cells is eightfold higher than
the cellular GABA content and can thus be used as an index of cellular
GAD activity. GABA release is 40% reduced by glucose (58 pmol/103 cells at 10 mM glucose vs. 94 pmol at 3 mM
glucose, P < 0.05). This suppressive effect of glucose
was not observed when glucose metabolism was blocked by mannoheptulose
or 2,4-dinitrophenol; it was amplified when ATP-dependent
-cell
activities were inhibited by addition of diazoxide, verapamil, or
cycloheximide or by reduction of extracellular calcium levels; it was
counteracted when
-cell functions were activated by nonmetabolized
agents, such as glibenclamide, IBMX, glucagon, or glucacon-like
peptide-1 (GLP-1), which are known to stimulate calcium-dependent
activities, such as hormone release and calcium-dependent ATPases.
These observations suggest that GABA release from
-cells varies with
the balance between ATP-producing and ATP-consuming activities in the
cells. Less GABA is released in conditions of elevated glucose
metabolism, and hence ATP production, but this effect is counteracted
by ATP-dependent activities. The notion that increased cytoplasmic ATP
levels can suppress GAD activity in
-cells, and hence GABA
production and release, is compatible with previous findings on ATP
suppression of brain GAD activity.
diabetes; glutamate decarboxylase; insulin
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INTRODUCTION |
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IDENTIFICATION OF ISLET
CELL ANTIBODIES that recognize glutamate decarboxylase (GAD) in
the pancreatic -cell (4, 5) has attracted attention to
the putative role of this protein in the development of the autoimmune
reactivity in type 1 diabetes (3). However, it is still
unclear which role the enzyme plays in
-cells under physiological
conditions. Pancreatic
-cells rank among the few nonneuronal cell
types that express GAD and contain its product,
-aminobutyric acid
(GABA) (6, 9, 12, 23, 27, 37, 38). They release GABA in
vitro in amounts that correspond to the cellular GABA production
(34). Locally released, GABA may act as a paracrine
inhibitor on adjacent glucagon- and/or somatostatin-producing islet
cells (6, 21, 29, 36). It may also serve as an autocrine
regulator of the
-cells, although this effect was not noticed in all
tested models (13, 14, 32). Inhibitory effects of GABA in
the endocrine pancreas are consistent with its well-known suppressive
actions as a neurotransmitter in the nervous system (16).
Electrophysiological measurements in guinea pig islet cells have
indicated the presence of a GABA-sensitive chloride channel
(28). We have previously shown that GABA release from
isolated rat
-cells is inhibited by high glucose levels (20 mM)
(34). In this study, we examine whether this suppressive effect of glucose is related to the increased metabolic activity of the cells or to their increased functional state. To this end, we
measured GABA release in the presence of inhibitors of either metabolic
or functional activities or after addition of nonmetabolic stimuli of
-cell functions.
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MATERIALS AND METHODS |
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Preparation and culture of rat islets and purified -cells.
Pancreatic islets were isolated from adult male Wistar rats by
collagenase digestion and then dissociated in calcium-free medium
containing trypsin and DNase (25). The dispersed islet cell preparation was then sorted in a FACStar Plus flow cytometer (Becton-Dickinson, Sunnyvale, CA). The single
-cells (>95% pure) were reaggregated during a 2-h shaking incubation at 37°C in Ham's F-10 medium (GIBCO, Strathclyde, UK) containing 2 mM glutamine, 10 mM
glucose, 2 mM calcium, 0.5 g/dl charcoal-extracted bovine serum albumin
(BSA) (type V, Boehringer Mannheim, Mannheim, Germany), 0.075 g/l
penicillin (Continental Pharma, Brussels, Belgium) and 0.1 g/l
streptomycin (Sigma, St. Louis, MO). The
-cell aggregates and islets
were cultured in suspension (5% CO2 humified air) in Sarstedt 24-well tissue culture plates at a concentration of 5 × 104
-cells/500 µl and 50 islets/500 µl of medium,
respectively. After a 16-h static preculture period, cells were
cultured for a second 24-h culture period in Ham's F-10 medium (GIBCO)
containing 2 mM glutamine, 0.5 g/dl charcoal-extracted BSA (type V,
Boehringer Mannheim), 0.075 g/l penicillin, and 0.1 g/l streptomycin
supplemented with one or more of the following test agents: 3-20
mM D-glucose, 0.3-2.0 mM calcium, 20 mM
D-mannoheptulose (Sigma), 100 µM 2,4-dinitrophenol (Sigma), 10 µM verapamil (Isoptine; Knoll, Ludwigshafen,
Germany), 200 µM diazoxide (ICN Biochemicals, Costa Mesa, CA), 5 µg/l cycloheximide (Sigma), 4 µM glibenclamide (ICN Biochemicals),
10 nM porcine glucagon (Novo, Bagsvaerd, Denmark), 10 nM glucagon-like
peptide-1-(7-36) amide (GLP-1; Sigma), 50 µM IBMX
(Janssen Chimica, Beerse, Belgium), and 100 µM
8-bromoadenosine-5',5'-cyclic monophosphorothioate, Rp-isomer
(Rp-8-BrcAMP; Biolog Life Science Institute, Bremen, Germany).
After this second culture period, cells and media were collected for
GABA and insulin determinations. The GABA and insulin cell contents
were determined in cell extracts after sonication.
Insulin, GABA, and GAD assays. The insulin radioimmunoassay was performed as previously described (17).
The chromatographic conditions and precolumn derivatization procedures for GABA determination have been previously reported (35). An isocratic, reverse-phase microbore liquid chromatography was used. Precolumn derivatization was performed with o-phthalaldehyde/tert-butylthiol and iodoacetamide. Before derivatization, culture medium and cell lysates were filtered through Ultrafree-MC 10 K filter units (Millipore, Bedford, MA). Recovery of GABA after standard addition exceeded 90%. Standard curves spanned the concentration range between 25 and 400 nM. The coefficients of variation for inter- and intra-assay imprecision were <5% (n = 20). GAD activity was measured as described by Smismans et al. (33) by use of the above-described HPLC method for GABA determination. Briefly, cell extracts were incubated for 1 h with 50 mM glutamate and in the presence or absence of 400 µM pyridoxal 5'-phosphate (PLP). GABA was measured at the start and after 1 h of incubation at 37°C, and the net increase was calculated as units of GAD/60 min. GAD expression was analyzed by Western blot analysis ofStatistical analysis.
Data are presented as means ± SE from 4 independent
experiments. The statistical significance between the experimental
conditions was calculated by two-tailed paired Student's
t-test. Correlations were evaluated by means of the Pearson
correlation coefficient.
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RESULTS |
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Comparison of GABA release from isolated islets and purified
-cells.
Isolated islets release GABA during culture. Over a 24-h period, the
amount of GABA released in the medium was eightfold higher than that
recovered in the islet tissue (Table 1).
At 10 mM glucose, GABA release was 20% lower (P < 0.05) than at 3 mM glucose; these lower extracellular levels were not
paralleled by a higher GABA content in the islet cells and therefore do
not result from a suppressed GABA secretion (Table 1). This suppressive
effect of glucose was also observed in purified
-cells, where it was more pronounced (Table 1, 40% reduction, P < 0.05)
except when 10
8 M glucagon was present; this suggests
that the interstitial glucagon concentrations in isolated islets
counteract the suppressive effect of glucose on GABA production. All
further studies were therefore conducted in purified
-cell
preparations, allowing us to examine the suppressive action of glucose
in the absence of possibly confounding influences by other cell types.
We selected the 6 and 20 mM glucose concentrations for these
experiments to compare the effects of near physiological and maximally
elevated glucose concentrations. All measurements were conducted after
2- or 24-h culture; under these conditions, the suppressive effects of
glucose on GABA release and production were reproduced (Fig.
1).
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GABA release during suppression of oxidative, synthetic, and
secretory activities.
The suppressive effect of 20 mM glucose on GABA release was not
detected in the presence of 20 mM mannoheptulose (Fig. 1), which is
known to block phosphorylation of glucose; this condition also
inhibited glucose-induced insulin release. Likewise, 2,4-dinitrophenol, known to block the more distal oxidative phosphorylation of the sugar,
also counteracted the suppressive effect of high glucose on GABA
release; because this agent can cause -cell necrosis within 1-3
days (31), its effect was investigated during a 2-h culture period. The choice of a shorter test period did not influence the suppressive effect of glucose on GABA release (Fig. 1).
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Effect of nonmetabolized stimuli of -cell functions.
Glibenclamide, glucagon, and GLP-1 are nonmetabolized stimuli of
-cell functions. Their addition to the 20 mM glucose medium did not
significantly increase insulin release over the 24-h culture period,
which can be attributed to the hyperactivated state of the
-cells
that are chronically exposed to this excessive glucose concentration
(17). When added to a 10 mM glucose medium,
10
8 M glucagon elevated insulin release by 84%
(10.2 ± 0.8 vs. 18.8 ± 2.4 ng/103
-cells,
P < 0.05). Irrespective of their effect on insulin
release, glibenclamide, glucagon, and GLP-1 counteracted the
suppressive effect of the 20 mM glucose culture on GABA release (Table
1 and Fig. 3). This was also the case
when IBMX was added to the 20 mM glucose medium (Fig. 3). This
phosphodiesterase inhibitor was tested at 0.3 mM calcium instead of 2 mM; at this lower calcium concentration, the suppressive effect of 20 mM glucose was more pronounced (Fig. 3). This IBMX effect on GABA
release was partly blocked by the protein kinase A inhibitor
Rp-8-BrcAMP (Fig. 3).
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Comparison of GABA content in the medium and cellular GABA and GAD65 content. In all tested conditions, the amount of GABA released in the medium during 24-h culture represented 90% of the total amount measured in medium plus cells. Therefore, this value can be used as an index for the GABA production during this period.
The observation of a reduced GABA production during culture at high glucose was not correlated with changes in GAD65 expression: no difference was noticed in Western blots of
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DISCUSSION |
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The present study confirms that the amount of GABA release from
rat -cells can be reduced by elevated glucose levels
(34). This glucose-induced suppression is dependent on
metabolization of the sugar, because it is not observed in the presence
of proximal or distal inhibitors of glucose metabolism, such as
mannoheptulose or 2,4-dinitrophenol. Glucose metabolism by the
-cells increases their ATP content (2, 11, 19). The
rise in total ATP is, however, small compared with the basal levels,
which probably represent, to a large extent, the ATP depots in cellular
organelles (15, 19). Measurement of free ATP levels in
cellular microdomains is more relevant to follow, but also more
difficult (15). It was recently shown that glucose
increases free ATP concentrations beneath the plasma membrane
(22). A local increase in ATP over ADP levels closes the
ATP-dependent potassium channels, which results in opening of L-type
calcium channels and stimulation of calcium-dependent processes such as
insulin release (1). Mannoheptulose and dinitrophenol
block glucose-induced ATP formation and thus glucose activation of a
number of
-cell functions (2, 11, 39). This raises the
question of whether the glucose suppression of GABA release is caused
by a rise in free ATP, by any of the other signals involved in the
stimulation of
-cell function, or by associated events.
A role of ATP in the regulation of GABA release should certainly be
considered in view of its well known influence on the activity of GAD
from other tissues. In the brain, GAD is activated by binding to its
cofactor PLP (10, 20, 26) and is then called holoGAD.
ApoGAD is the PLP-free form that is stabilized by ATP. -Cells
exposed to high glucose may thus present an increase in free ATP in the
vicinity of GAD, thus reducing this enzyme's activity and GABA
production. The existence of such a mechanism is, however, difficult to
investigate because local variations in free ATP levels and in GAD
activity cannot yet be monitored in compartments of intact
-cells.
We compared the expression of the GAD65 protein and the GAD activity in
extracts of
-cells that had been cultured for 24 h at basal or
high glucose, and we found no differences. In both conditions, the
enzymatic activity was three- to fourfold higher when measured in the
presence of PLP.
-Cell extracts thus exhibit a smaller fraction of
GAD that is active without PLP and a larger fraction that is activated by PLP. Activation by PLP is compatible with a role of ATP in stabilizing the enzyme in its inactive apoform (20, 26).
Measurement of GAD activities in cell extracts are, however, unlikely
to reflect ATP-induced variations that might have occurred in the
vicinity of GAD within living cells. They will be influenced by other
factors, such as ATP released from mitochondria and secretory vesicles after rupture of cellular membranes.
Our data do not support the possibility that the glucose-induced suppression of GABA release results from the sugar's activation of the cellular synthetic or secretory activities. Indeed, a block of protein synthesis by cycloheximide did not interfere with the suppressive effect of glucose; on the contrary, the glucose effect was amplified, as if inhibition of this ATP-consuming function (30) had resulted in a higher ATP suppression of GAD activity. This hypothesis is also supported by observations in the presence of inhibitors of insulin release, another ATP-consuming process.
The suppressive effect of glucose on GABA release was not mimicked by
nonmetabolized stimuli of -cell functions. In fact, the tested
stimuli counteracted the glucose effect. This was the case for
glibenclamide, which is known to increase the calcium signal
(1), as well as for IBMX, glucagon, and GLP-1, which increase cellular cAMP levels and calcium translocation (1, 24). These stimuli may in fact reduce subcellular ATP levels through increased ATP consumption by their stimulation of the secretory
activity. Although this explanation would further support the view that
local decreases in ATP are associated with an increased GABA production
and release, the present experimental conditions allow only in part
such extrapolation. In one set of experiments, addition of glucagon
(Table 1) and IBMX (Fig. 3) indeed markedly increased insulin release
in parallel to a counteraction of the suppressive effect of glucose on
GABA production; this is compatible with the idea that their
ATP-consuming effect reduces the ATP stabilization of inactive GAD and,
hence, results in higher GABA production. However, in another set of
experiments, glibenclamide, glucagon, and GLP-1 exerted a similar
effect on GABA production without significantly increasing insulin
release (Fig. 3). Interestingly, these two sets of experiments differed
in the prevailing calcium concentration (0.3 mM in the first set and 2 mM in the second). We also noticed that the suppressive effect of
glucose on GABA production and release was much more marked at lower
calcium concentration. Calcium thus exerts a major influence on GABA
production; this can be explained by its permissive role in ATP
consumption, be it under the form of increasing insulin release or of
activating the calcium ATPases at the endoplasmic reticulum. Previous
work has indeed shown that calcium suppresses the glucose-induced rise in islet ATP levels (39). This effect can be achieved by
increasing extracellular calcium levels and/or adding agents such as
glibenclamide, glucagon, GLP-1, or IBMX, which are known to increase
intracellular calcium concentrations. Glibenclamide can also reduce
glucose-induced ATP levels through its action on the
Na-K+-ATPase (7, 8). That these same
conditions counteract the glucose-induced suppression on GABA
production and release is thus compatible with the concept that an
increase or decrease in local ATP levels results in, respectively, a
reduced or elevated GAD activity and GABA production and release.
In conclusion, our data indicate that the suppressive effect of glucose
on GABA release depends on its metabolism, but not on the associated
increases in secretory or synthetic activities. It is not mimicked by
nonmetabolized stimuli of -cell functions but is instead
counteracted by such agents, conceivably through their activation of
ATP-consuming processes, such as hormone release and Ca-dependent
ATPases. We propose that an increase in free ATP levels can suppress
GAD activity in
-cells, which would be compatible with previous
observations on brain GAD. Further investigations on local ATP levels
in
-cells should test this hypothesis.
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ACKNOWLEDGEMENTS |
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We thank Drs. Michotte, Sarre, and Smolders (Farmaceutisch Instituut, Vrije Universiteit Brussel) for help in HPLC determinations and J.-C. Hannaert, G. Schoonjans, G. Stangé, C. De Ryck, and R. Berckmans for technical assistance.
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FOOTNOTES |
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The present work was supported by the Belgian Fonds voor Wetenschappelijk Onderzoek (Grant G.0376.97 and a research fellowship to F. Winnock and S. Dejonghe) and by grants from the services of the Prime Minister (Interuniversity Attraction Pole P4/21).
Address for reprint requests and other correspondence: D. Pipeleers, Diabetes Research Center, Vrije Univiversiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium (E-mail: Daniel.Pipeleers{at}vub.ac.be).
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.
10.1152/ajpendo.00071.2001
Received 2 March 2001; accepted in final form 29 November 2001.
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