(Received for publication, August 23, 1995)
From the
To determine if endogenous -aminobutyric acid (GABA) is
secreted by a pancreatic
-cell-derived cell line and to determine
the effects of glucose on GABA release,
TC6 cultures were
incubated in the presence of 1 or 10 mmol/l glucose for 12 h and then
subjected to a 2-h secretion test in Krebs-Ringer buffer containing 1
or 10 mmol/l glucose.
TC6-conditioned medium was collected at 15,
30, 60, and 120 min after glucose stimulation for GABA analysis by high
pressure liquid chromatography-electrochemical detection. After 30 min,
medium GABA concentrations were significantly higher (p <
0.05) in cultures that were exposed to high glucose during both the
12-h incubation period and the 2-h secretion test than in the remaining
three glucose combinations. To address possible roles of
-cell-derived GABA, the effect of GABA on glucagon secretion from
pancreatic
TC6 cells was tested at concentrations released from
TC6 cells. Inhibition of glucagon secretion by
TC6 cells was
observed in the presence of GABA at concentrations equivalent to
concentrations secreted by
TC6 cells. The inhibitory effects of
GABA on glucagon secretion by
TC6 cells were blocked by the
GABA
receptor antagonist bicuculline and were dissociated
from the inhibitory effects of glucose. Together, these results provide
the first documentation that endogenous GABA is released from a highly
differentiated
-cell line and that glucose and GABA independently
attenuate glucagon secretion by a pancreatic
-cell line.
Glutamic acid decarboxylase (L-glutamate 1-carboxylase;
EC 4.1.1.15) has been identified as an early target antigen of the
T-lymphocyte-mediated destruction of pancreatic -cells causing
insulin-dependent diabetes mellitus(1, 2) .
Development of insulitis and diabetes was prevented in NOD mice by
inactivating glutamic acid decarboxylase-reactive
T-cells(1, 2) , and serum antibodies to glutamic acid
decarboxylase are predictive of prediabetes in humans(3) .
Recent focus on glutamic acid decarboxylase's role in
insulin-dependent diabetes mellitus has exposed a striking lack of
knowledge as to the functional relevance of glutamic acid decarboxylase
and its synthetic product,
-aminobutyric acid (GABA), (
)in the endocrine pancreas.
Briel and associates (4) and Okada and associates (5) were among the first
to report that GABA was present in the endocrine pancreas at
concentrations comparable with those found in the central nervous
system. There is limited GABAergic innervation of the acinar pancreas,
and GABA-containing neuronal processes are located adjacent to islets,
but few neuronal processes are found within an islet(5) . The
highest concentrations of GABA in the pancreas are found within
endocrine islets, with up to 10-fold higher concentrations than in the
exocrine acinar pancreas(6) . Within pancreatic islets, GABA is
confined to -cells as are the two enzymes responsible for GABA
synthesis and metabolism, glutamic acid decarboxylase and GABA
transaminase (GABA-
-ketoglutarate transaminase, EC 2.6.1.19
(reviewed in Refs. 7 and 8). Within isolated
-cells and
TC3
cells, GABA is found both in the cytoplasm and in distinct
synaptic-like microvesicles(9, 10, 11) ,
which is suggestive of regulated release; however, neither constitutive
nor regulated release of GABA from purified
-cells has been
demonstrated.
The role of GABA in islet function is also unclear.
GABA receptors have been demonstrated on both pancreatic -cells
and
-cells but not on
-cells(12) . In vitro studies with isolated islets and with perfused pancreas of mice,
rats, and dogs (8, 12, 13, 14) support the idea
that
-cell GABA may modulate
-cell function in a paracrine
manner, although data from purified cells have not been reported. In
the present study, differentiated pancreatic
- and
-cell
lines derived from transgenic mice (15, 16) were used
to determine (i) if GABA is released from transformed
-cells, (ii)
if GABA secretion is influenced by extracellular glucose
concentrations, and (iii) to test the effects of GABA on glucagon
secretion from a purified
-cell line.
The TC6 cell line was derived by a similar approach from a
glucagonoma created in transgenic mice expressing the SV40 large
T-antigen oncogene under the control of the rat preproglucagon promoter (15) . Glucagon-secreting
TC6 cells were selectively
cloned from the original
TC1 cell line by the limiting dilution
method(17) . These cells maintain many of the differentiated
characteristics of
-cells in situ including the synthesis
and secretion of glucagon and not insulin(17, 18) .
TC6 cells were kindly provided by Dr. Edward H. Leiter, The
Jackson Laboratory.
The cell lines were routinely maintained in
Dulbecco's minimal essential medium (DMEM) supplemented to a
final glucose concentration of 25 mmol/l with Eagle's minimal
essential medium nonessential amino acids, 44 mmol/l sodium
bicarbonate, 15 mmol/l HEPES, 50 mg/l gentamicin sulfate, 15% horse
serum, and 2.5% FetalClone II (HyClone Laboratories, Inc., Logan, UT).
Unless otherwise specified, cell culture reagents were purchased from
Life Technologies, Inc. Cells were seeded at a density of 5
10
cells/well into 24-well plates (Corning, Corning, NY).
Cultures were maintained in a humidified atmosphere of 95% air, 5%
CO
at 37 °C. Experiments were performed when cells were
approximately 70% confluent (approximately 4
10
cells/well). Following the culture period, cells were harvested
and sonicated in 0.5 ml of phosphate-buffered saline, 0.1% Triton X-100
(Fisher Biotech, Fair Lawn, NJ). Samples were stored at -20°C
for later protein determination by the Bradford microassay method
(Bio-Rad).
GABA
dose-response studies were conducted to determine the effects of GABA
on glucagon secretion by TC6 cells (passage 28). The GABA
concentrations tested were in the same molar range as those secreted by
TC6 cells. On the day of the experiment, medium was removed and
TC6 cells were washed twice with KRB (10 mmol/l glucose). Cells
were then preincubated at 37 °C for 1 h in KRB containing 10 mmol/l
glucose, followed by incubation in fresh KRB containing 1 mmol/l
glucose alone or with increasing GABA concentrations (50-200
nmol/l GABA) or with 10 mmol/l glucose plus 100 nmol/l GABA for 2 h.
Cells exposed to 1 or 10 mmol/l glucose plus 100 nmol/l GABA were also
treated with bicuculline (100 nmol/l), a specific competitive
antagonist of the GABA
receptor(19) . At the end of
secretion test,
TC6-conditioned medium was collected for later
glucagon determination by RIA. Cells were seeded equally among wells;
protein values per well were similar across glucose, GABA, and
bicuculline treatments at the end of the culture period. Data are
expressed as percent of control (1 mmol/l glucose).
Glucagon
concentrations in TC6-conditioned medium were determined by double
antibody RIA using a published assay that has been validated in our
laboratory(24) . Porcine/bovine glucagon was used as a
standard. Standards, antibodies, and
I-glucagon were
obtained from Linco Research, Inc., St. Louis, MO. The inter- and
intraassay coefficients of variation were both less than 5%.
Figure 1:
Effect of glucose on GABA release from
TC6 cultures.
TC6 cells were first exposed to either 1 mmol/l (open symbols) or 10 mmol/l (closed symbols) glucose
during a 12-h incubation period. The amount of GABA released by
TC6 cells in response to 1 mmol/l (diamonds) and 10
mmol/l (squares) glucose in HEPES-buffered KRB was then
determined during a subsequent 2-h secretion test. Fifty-microliter
aliquots of
TC6-conditioned KRB, collected at intervals of 15, 30,
60, and 120 min, were assayed for GABA content by HPLC-electrochemical
detection. Data are presented as nmol/l and represent the mean ±
S.E. from triplicate wells for glucose treatment for each time period. Asterisks indicate significant (p < 0.05) glucose
effects within time.
Figure 2:
Effect of GABA on glucagon secretion by
TC6 cells.
TC6 cultures were exposed to KRB supplemented with
1 mmol/l glucose alone or with increasing GABA concentrations
(50-200 nmol/l GABA) or with 10 mmol/l glucose plus 100 nmol/l
GABA for 2 h. At the end of the secretion test,
TC6-conditioned
medium was collected for later glucagon determination by RIA. Data
represent the mean ± S.E. from triplicate wells from a
representative experiment. Glucagon secretion is expressed as a percent
of control (1 mmol/l glucose without GABA). Asterisks indicate
that the values are significantly (p < 0.05) less than the
values obtained from control cultures (1 mmol/l glucose without GABA). Dark shaded bars, -GABA; light shaded bars, +GABA.
Figure 3:
Effect of glucose, GABA, and bicuculline,
a GABA receptor antagonist, on glucagon secretion by
TC6 cells.
TC6 cultures were exposed to KRB supplemented with
1 or 10 mmol/l glucose alone or with 100 nmol/l GABA plus or minus 100
nmol/l bicuculline for 2 h. At the end of secretion test,
TC6-conditioned medium was collected for later glucagon
determination by RIA. Data represent the mean ± S.E. from two
experiments. Glucagon secretion is expressed as a percent of control (1
mmol/l glucose without GABA). Asterisks indicate that the
values are significantly (p < 0.05) less than the values
obtained from control cultures (1 mmol/l glucose without GABA). Dark shaded bars, -GABA; light shaded bars, +GABA (100 nM); striped bars, +GABA
(100 nM) + bicuculline
(100nM).
To test the
responsiveness and sensitivity of TC6 cells to GABA,
TC6
cultures were incubated for 1 h in the presence of 10 mmol/l glucose
and then switched to fresh KRB containing 1 mmol/l glucose and
increasing GABA concentrations in a range of concentrations
(50-200 nmol/l GABA) determined earlier to be secreted by
TC6 cells. Glucagon secretion by
TC6 cultures exposed to 1
mmol/l glucose was inhibited (p < 0.05) by GABA at
concentrations equal to and greater than 100 nmol/l (Fig. 2).
These results demonstrate that GABA inhibition of glucagon secretion by
-cells occurs at concentrations secreted by
-cells. GABA (100
nmol/l) did not decrease glucagon secretion by
TC6 cells cultured
in high (10 mmol/l) glucose during the 2-h secretion test ( Fig. 2and Fig. 3).
To test the specificity of
GABA's inhibitory effects on -cell function, glucagon
concentrations were compared in glucose-stimulated
TC6 cultures
incubated in the presence of both GABA (100 nmol/l) and bicuculline
(100 nmol/l), a GABA
receptor antagonist(19) . The
inhibitory effect (p < 0.05) of GABA on glucagon secretion
by
TC6 cells in response to 1 mmol/l glucose was abolished in the
presence of bicuculline (Fig. 3). A slight decrease in glucagon
secretion was observed when GABA was added to
TC6 cultures exposed
to 10 mmol/l glucose ( Fig. 2and Fig. 3). This
GABA-mediated decrease was also abolished in response to bicuculline (Fig. 3).
The present results provide the first documentation that
endogenous GABA is secreted by a pancreatic -cell line and that
release is influenced by extracellular glucose concentrations. The
results further provide evidence that GABA, at concentrations similar
to those released from
-cells, will attenuate glucagon release
from pancreatic
-cells. While previous studies (reviewed in (8) ) have demonstrated GABA synthesis by isolated islets and
GABA release from perfused pancreas, the source of GABA was not
identified conclusively in either case. Using a sensitive HPLC
procedure similar to the one employed here, Thomas-Reetz and associates (10) did not detect GABA peaks after stimulation of
TC3
cells with various insulin secretagogues, including glucose. The
presence of amino acids in cell culture media limits GABA detection by
HPLC, which led us to use KRB routinely in GABA secretion studies. This
together with our use of the more differentiated
TC6 cell line may
explain why we were able to measure GABA release from the
-cell
line.
A positive relationship between glucose concentration and GABA
secretion by TC6 cells was observed in the present study. Few
studies investigating glucose modulation of either GABA synthesis or
release have been reported in the literature. Gylfe and Hellman (25) reported that GABA concentrations in freeze-dried islet
sections and in fresh islet extracts from starved ob/ob mice (background strain not designated) were numerically higher
than in preparations from fed mice. The mice were given free access to
food or were fasted for 3 days prior to islet isolation. While those
results indicate that GABA synthesis was modulated by presumed distinct
states of glycemia, neither blood concentrations of glucose nor the
islet hormones were provided as conformation. Regarding release, Gerber
and Hare (26) reported that external concentrations of GABA
doubled within 15 min when pieces of rabbit pancreas were transferred
from a buffer containing 2.8 mmol/l glucose to one containing 27.8
mmol/l glucose.
GABA concentrations were highest in TC6
cultures exposed to high glucose during both the 12-h incubation period
and the subsequent 2-h secretion test in the present study (Fig. 1). Thus, external glucose may influence the amount of
GABA released in two ways. First, chronic glucose levels may influence
GABA biosynthesis, and second, GABA release may be dependent on a
glucose-stimulated membrane depolarization event. That preproinsulin
biosynthesis and insulin secretion are stimulated by distinct
glucose-activated events provides precedent for more than one
glucose-sensitive pathway being operative in pancreatic
-cells(27, 28) . The observation that GABA
concentrations did not differ between
TC6 treatment groups exposed
to low glucose during the 12-h incubation period but differently
stimulated with low or high glucose during the 2-h secretion test
supports the postulate that glucose availability influences GABA
synthesis by
-cells. A preliminary comparison of GABA
concentrations in
TC6-conditioned medium after 2- or 12-h
incubation periods in 1 or 10 mM glucose demonstrated that
GABA concentrations were higher after a longer period of incubation for
both glucose concentrations (not shown). Moreover, as demonstrated by
the present results, the amount of GABA released during a subsequent
secretion test was dependent on the glucose concentration present
during the incubation period. Notably, Gylfe and Hellman (25) demonstrated that GABA can be synthesized from glucose in
islets. In the brain, estimates are that 10-40% of GABA in
neurons is derived from glucose. Michalik and Erecinska (8) offered the view that GABA may provide fuel for the
generation of ATP, via the GABA shunt, in islets when glucose is
deficient. The present demonstration that GABA is synthesized and
secreted by
TC6 cells should facilitate future efforts to define
the extent of
-cell GABA metabolism using these cells as a model
system.
GABA secretion from TC6 cells reflected extracellular
glucose concentrations; however, insulin release did not differ between
the low and high glucose treatments. The insulin response noted is
consistent with previous reports of diminished glucose responsiveness
in the murine
-cell lines with propagation in
culture(16, 29, 30) . The shift in glucose
sensitivity in these cell lines has been associated with an increase in
hexokinase activity(16) . The present demonstration that GABA
secretion, but not insulin secretion, remained responsive to
differences in extracellular glucose in passage 17
TC6 cells
indicates that glucose-regulated pathways modulating GABA secretion may
be more sensitive to or otherwise distinct from those that regulate
insulin secretion. Clearly, additional studies are required to
precisely define the glucose-sensitive pathways that modulate GABA
biosynthesis and release by
-cells.
The role of locally
produced GABA is also unresolved. The possibility that GABA released
from -cells may have a paracrine role in the islet is proposed
frequently(7, 8, 31, 32) . Secretion
of GABA from
TC6 cells prompted us to directly test the modulatory
effects of GABA on glucagon secretion by
TC6 cells, a
glucose-sensitive glucagon-producing
-cell line(15) . The
results provide clear indication that (i) GABA attenuates glucagon
release from
TC6 cells; (ii) GABA is effective at concentrations
that are released from
TC6 cells; (iii) GABA and glucose
independently inhibit glucagon secretion by
TC6 cells; and (iv)
that GABA's inhibitory effects on glucagon secretion are mediated
through GABA
receptors. Previous studies demonstrating GABA
inhibition of glucagon secretion in islets used supraphysiological
concentrations of GABA (µM range(12, 33) ).
Although it is not possible
to infer an in vivo role of GABA from these results, the
observations do provide clear evidence that GABA and glucose can
inhibit glucagon release independently and thereby argue against the
proposal that the inhibitory effects of glucose on glucagon are
mediated by -cell GABA(12) . Having
-cells
independently responsive to both glucose and GABA may play a key role
in the physiological control of blood glucose. Initially the short term
rise in blood glucose associated with meal ingestion might inhibit
glucagon secretion directly, while indirect inhibition through
glucose-stimulated GABA release from neighboring
-cells may become
important for tonic suppression of glucagon as normoglycemia is
restored. The present observation that glucose history influences the amount of GABA released by
TC6 cells agrees
with that possibility. A complete understanding of the role of GABA in
physiological regulation of glucose will require investigation of
strict temporal parameters that can only be addressed in vivo.
A clear impetus for such studies is established by the present
demonstration that GABA is secreted by a
-cell line and that the
effects of glucose and GABA on glucagon secretion can be dissociated
with a defined model system.