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Diabetes 56:320-327, 2007
DOI: 10.2337/db06-0712
© 2007 by the American Diabetes Association
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Glucose-Dependent Regulation of {gamma}-Aminobutyric Acid (GABAA) Receptor Expression in Mouse Pancreatic Islet {alpha}-Cells

Sarah J. Bailey, Magalie A. Ravier, and Guy A. Rutter

Henry Wellcome Laboratories for Integrated Cell Signalling and Department of Biochemistry, School of Medical Sciences, University of Bristol, U.K. The current affiliation for S.J.B. is the Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath, U.K. The current affiliation for G.A.R. is the Department of Cell Biology, Division of Medicine, Faculty of Medicine, Imperial College, Sir London, U.K

Address correspondence and reprint requests to Professor Guy A. Rutter, Department of Cell Biology, Division of Medicine, Faculty of Medicine, Imperial College, Sir Alexander Fleming Building, Exhibition Road, London SW7 2AZ, U.K. E-mail: g.rutter{at}imperial.ac.uk. Or to Dr. Sarah Bailey, Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY, U.K. E-mail: s.bailey{at}bath.ac.uk

Abbreviations: CREB, cAMP response element binding protein; GABA, {gamma}-aminobutyric acid; GABAAR, GABAA receptor; PPG, preproglucagon; mRFP, monomeric red fluorescent protein


   ABSTRACT
TOP
 ABSTRACT
RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The mechanism(s) by which glucose regulates glucagon secretion both acutely and in the longer term remain unclear. Added to isolated mouse islets in the presence of 0.5 mmol/l glucose, {gamma}-aminobutyric acid (GABA) inhibited glucagon release to a similar extent (46%) as 10 mmol/l glucose (55%), and the selective GABAA receptor (GABAAR) antagonist SR95531 substantially reversed the inhibition of glucagon release by high glucose. GABAAR {alpha}4, ß3, and {gamma}2 subunit mRNAs were detected in mouse islets and clonal {alpha}TC1-9 cells, and immunocytochemistry confirmed the presence of GABAARs at the plasma membrane of primary {alpha}-cells. Glucose dose-dependently increased GABAAR expression in both islets and {alpha}TC1-9 cells such that mRNA levels at 16 mmol/l glucose were ~3.0-fold ({alpha}4), 2.0-fold (ß3), or 1.5-fold ({gamma}2) higher than at basal glucose concentrations (2.5 or 1.0 mmol/l, respectively). These effects were mimicked by depolarizing concentrations of K+ and reversed by the L-type Ca2+ channel blocker nimodipine. We conclude that 1) release of GABA from neighboring ß-cells contributes substantially to the acute inhibition of glucagon secretion from mouse islets by glucose and 2) that changes in GABAAR expression, mediated by changes in intracellular free Ca2+ concentration, may modulate this response in the long term.

Although glucose is a well-defined regulator of glucagon secretion (1), understanding of the molecular mechanisms through which the sugar acts on pancreatic {alpha}-cells remains fragmentary. It is generally accepted that these cells are electrically active at low glucose concentrations (2) and silent at high glucose concentrations (3) and that the consequent lowering of cytosolic free Ca2+ concentrations as glucose concentrations rise underlies the diminished glucagon release (4,5). Nevertheless, the respective roles in mediating the effects of glucose of 1) a direct metabolic effect of the sugar on individual {alpha} -cells, 2) the secretion of paracrine factors from neighboring ß-cells or other islets cells, and 3) autonomic inputs (6) are still disputed and may be highly species specific.

We have recently shown that, in isolated mouse {alpha}-cells, glucose exerts a direct inhibitory effect on {alpha}-cell activity (5), in contrast to a reported activatory effect of glucose on isolated rat {alpha}-cells (7). Moreover, release from ß-cells of the potential paracrine factors insulin (8) and Zn2+ ions, which may be important in the rat (9,10), appears not to be involved in the mouse (5). At present, however, the relative contribution of the direct effect of glucose to the overall action of glucose on mouse {alpha}-cells is undefined.

{gamma}-Aminobutyric acid (GABA), the major inhibitory transmitter in the central nervous system, has long been known to be present at significant concentrations in the endocrine pancreas (11–13). Furthermore, the presence of GAD and GABA transaminase has also been demonstrated in ß-cells, indicating local synthesis and metabolism of GABA (14). Moreover, GABA appears to be released from rat pancreatic ß-cells in response to depolarization-induced increases in intracellular free [Ca2+] (15). Whether GABA release from ß-cells contributes significantly to the effects of glucose on glucagon secretion remains contested. Thus, exogenous GABA had no effect on glucagon release from the isolated dog pancreas (16) and, whether administered orally or intravenously, GABA failed to alter circulating glucagon levels in vivo in this species (17). In humans, oral GABA increased circulating levels of glucagon (18,19). By contrast, added to isolated mouse islets at 0 mmol/l glucose, exogenous GABA inhibited glucagon secretion by 17% (compared with the 57% inhibition elicited by high glucose concentrations) (20) and by 28% the release of glucagon from guinea pig islets stimulated with arginine (21). Furthermore, blockade of GABA receptors diminished the inhibitory effects of glucose partially in guinea pig (21) and more completely in rat islets (22), and insulin was recently reported to induce GABA receptor trafficking to the plasma membrane (23). Finally, GABA receptor blockade with bicuculline slightly potentiated the action of glucose on glucagon secretion from mouse islets (20), arguing against a significant controlling role for endogenous GABA in this species.

Ionotropic GABAA receptors (GABAARs) are multi-subunit ligand-gated chloride ion channels. At least 19 related GABAAR subunits have been identified in the central nervous system (6{alpha}, 4ß, 3{gamma}, 1{delta}, 1{varepsilon}, 1{pi}, and 3{rho} subunits) (24). GABAAR assembly is restricted and proposed to produce pentameric receptors that are combinations of at least 1{alpha} and 1ß, with a typical stoichiometry of 2, 2, and 1 (25). GABAAR heterogeneity confers a complex pharmacology and distinct functional properties on the channels (26). A feature of GABAAR expression in the central nervous system is its plasticity, with changes during development (27) as well as in models of premenstrual syndrome (28), focal ischemia (29), and epilepsy (30).

The presence of several GABAAR subunits has been described in guinea pig (21), human ({alpha}2, ß3, {gamma}1) (31), and rat ({alpha}1, 4; ß1, 2, 3 [22], {alpha}1, 2, 3; ß1, 2, 3; {gamma}1, 2; {delta} [32], and {alpha}1, 2; ß1; {gamma}2 [33]) pancreatic tissue as well as in insulinoma cell lines (33–35), though in only a few instances has cell surface expression of GABAAR subunit protein been demonstrated. However, it is not known whether pancreatic GABAARs are subject to the same plasticity of expression seen in neuronal GABAARs. Our aims here were 1) to investigate the extent to which GABA release plays a role in glucose-induced suppression of glucagon release in mouse islets, using the selective GABAAR antagonist SR95531 (22); 2) to determine whether GABAAR subunit expression is regulated by glucose in {alpha} -cells; and, if so, 3) to explore the molecular mechanisms involved.


   RESEARCH DESIGN AND METHODS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Islet isolation and culture.
Collagenase digestion was used to isolate intact islets from the pancreas of adult female CD1 mice (12–16 weeks) before culture in RPMI 1640 medium (10 mmol/l glucose) overnight before experiments (5). {alpha}TC1-9 cells (passage number 40–43; American Type Culture Collection) were grown in Dulbecco’s modified Eagle’s medium containing 16 mmol/l glucose (5). For measurement of GABAAR expression, cells were cultured in 16 mmol/l glucose to 60% confluence and switched to the test condition for 24 h.

Generation of preproglucagon–mRFP adenovirus (PPG-mRFP).
To identify living islet {alpha}-cells, using an analogous approach to that previously used to monitor Ca2+ in these cells (5), an adenovirus encoding monomeric red fluorescent protein (mRFP) under the control of the preproglucagon (PPG) (1.6 Kb) promoter was generated. Luciferase cDNA was removed from pShuttle-PPG-Luc (5) by digestion with HindIII and XbaI. cDNA encoding mRFP was then ligated into the same HindIII XbaI site of pShuttle-PPG-Luc to generate pPPG-mRFP. Adenoviruses were prepared as shown by Ainscow and Rutter (36).

Glucagon secretion.
Islets (second day of culture) and {alpha}TC1-9 cells were preincubated for 1 h at 37°C in Krebs buffer containing (in mmol/l) 120 NaCl, 4.8 KCl, 1.5 CaCl2, 1.2 MgCl2, 24 NaHCO3, 10 glucose, and 1 mg/ml BSA at pH 7.4. {alpha}TC1-9 cells in 12-well plates were incubated for 40 min in 700 µl Krebs buffer adjusted for each test condition. Islets were handpicked in groups of seven to eight and incubated for 1 h in 400 µl Krebs buffer. All medium was collected after the incubation, and the cells were lysed in 500 µl acidified ethanol/0.1% triton. Glucagon was assayed in medium and cell contents by radioimmunoassay (Linco Research, St. Charles, MO).

RNA isolation and RT-PCR.
Total RNA was isolated from {alpha}TC1-9 cells and islets using TRIzol reagent (Invitrogen Technologies). Total RNA from mouse brain was used as a positive control for GABAAR expression. One-step reverse transcription PCR (RT-PCR) was performed using Superscript One-Step RTPCR system with Platinum TaqDNA Polymerase (Invitrogen). Published gene-specific primer sequences for GABAAR {alpha}4 (37); {alpha}1, 2, 3, 5, 6; ß1–3; {gamma}1–3; and {delta} (38) subunits were used. Additional primers were used for {alpha}1 (5'-TTTCGGACCAGTTTCAGACC-3'; 5'-AACGTGACCCATCTTCTGCT-3'; 396 bp) and {varepsilon} (5'-CGGTTTGGCTTCATTGTCTT-3'; 5'-AGAAGTCCAAAGCCGTGAGA-3'; 202 bp). PCR amplifications were performed under standard conditions, and the identity of all amplicons was expressed in {alpha}TC and islet cells confirmed by DNA sequencing. PCR products were electrophoresed on 1% agarose gels with GeneRuler 100 bp DNA Ladder Plus (MBI Fermentas).

Quantitative real-time RT-PCR.
Quantitative analysis of the abundance of GABAAR {alpha}4, ß3, and {gamma}2 subunit mRNAs was performed on a DNA Engine Opticon System (MJ Research). Reverse transcription was performed using random hexamers with Omniscript reverse transcriptase (Qiagen), and real-time PCR detection used Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen). The reference was18S rRNA (39), and gene-specific primers for GABAARs were designed using GenBank sequences (Gabra4 [200 bp]: 5'-TGTCACCAGCTTTGGGCCCGTT-3'; 5'-GGAGCTGTCATGTTATGTGAGAC-3'; Gabrb3 (201 bp): 5'-ATTACCACCGTGCTCACCAT-3'; 5'-TGTCTTCTCCGCAAGCTTCT-3'; Gabrg2 (91 bp): 5'-GGAGCCTGGAGACATGGGA-3'; 5'-TGAACAAGCAAAAGGCGGTA-3'). The identity of each amplicon and specificity of amplification was confirmed by gel electrophoresis and melting curve analysis. All primer combinations were optimized and validated for relative quantification of gene expression using the comparative threshold cycle method (39) described in the PE Applied Biosystems sequence detection system user bulletin #2 (http://www.appliedbiosystems.com). Briefly, data for each target gene were normalized to the endogenous control gene (rRNA), and the fold change in target gene mRNA abundance was determined using the 2–{Delta}{Delta}Ct method such that levels in treated condition are expressed relative to control (=1) (40).

Immunocytochemistry.
Freshly isolated islets were dispersed to single cells by mechanical dissociation in calcium-free medium. Single cells were cultured overnight and infected with the pPPG-mRFP adenovirus (6 h) and cultured for a further 36 h before labeling. Cells were fixed in 4% paraformaldehyde, permeabilized in 0.1% Triton X-100, incubated with primary antibodies to GABAAR {alpha}4 (1:50; Santa Cruz Biotechnology) or {gamma}2 (1:50; Calbiochem) subunits overnight at 4°C, and detected with fluorescein isothiocyanate–anti-rabbit IgG (1:500; Jackson ImmunoResearch Laboratories). Images were captured on a Leica TCS NT laser scanning confocal microscope using a 63x oil immersion objective.

Semiquantitative Western (immuno) blot analysis.
{alpha}TC1-9 cells were homogenized in buffer containing 10 mmol/l Tris, pH 7.4, 0.32 mol/l sucrose, 5 mmol/l EDTA, and protease inhibitor cocktail (Complete Mini; Roche). Aliquots of crude membrane samples were assayed for protein content using BCA protein assay (Pierce), and increasing amounts of protein (10–40 µg) were separated by SDS-PAGE on 10% (wt/vol) polyacrylamide gels. Proteins were transferred onto polyvinylidene difluoride membranes (Millipore). The blots were blocked for 2 h in Tris-buffered saline/5% nonfat dry milk before incubation with the GABAAR {alpha}4 subunit antibody (1:100; Santa Cruz Biotechnologies) for 1 h at room temperature. Horseradish peroxidase–conjugated donkey anti-rabbit (1:7,500; Amersham Biosciences) and Supersignal West Pico Chemiluminescent Substrate (Pierce) were used for chemiluminescent detection. To ensure an analysis in the linear range, films were exposed for various times, and the quantification of immunoreactive bands was performed using the National Institutes of Health ImageJ program (http://rsb.info.nih.gov/ij).

Data analysis.
Data are expressed as the mean ± SE for the number of experiments indicated. Statistical significance and differences between means were evaluated using the Student’s t test.


   RESULTS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Role of GABA in glucose-regulated glucagon secretion.
The effects of GABA on glucagon secretion from mouse islets or {alpha}TC1-9 cells are shown in Fig. 1. As expected (1), glucose dose-dependently inhibited glucagon secretion in both cases, with near-maximal inhibition of glucagon secretion in islets achieved at ~5 mmol/l glucose (46.3 ± 7.8% of 0.5 mmol/l glucose value, n = 5, P < 0.05) and in {alpha}TC1-9 cells at ~1 mmol/l glucose (40.5 ± 5.1% of 0 mmol/l glucose value, n = 4, P < 0.05) (5). At low glucose concentrations, at which glucagon secretion is stimulated, GABA (10 or 100 µmol/l) significantly reduced glucagon secretion (Fig. 1A and B) from both islets (62.1 ± 11.4 or 54.1 ± 5.4% of control, n = 4, P < 0.05) or {alpha}TC1-9 cells (60.8 ± 11.6 or 42.7 ± 11.0% of control, n = 4, P < 0.05), while exerting no effect at high glucose concentrations.


Figure 1
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  		FIG. 1. GABA, acting via GABAARs, inhibited glucagon secretion in mouse pancreatic {alpha}-cells. Glucose-dependent suppression of glucagon secretion was measured in the absence ({circ}) and presence of 10 µmol/l GABA (•) and 100 µmol/l ({diamondsuit}) in intact mouse islets (A) and in {alpha}TC1-9 cells (B). The effects of the GABAAR antagonist SR95531 (10 µmol/l, SR) on GABA-mediated (10 µmol/l) inhibition of glucagon secretion in intact mouse islets (C and E) and {alpha}TC1-9 cells (D and F) are shown. Data are means ± SE; n = 4–7; *P < 0.05. Con, control.

 
To investigate the role of GABAARs, we next examined the effects of the selective GABAAR antagonist, SR95531 (22), on the ability of GABA to suppress glucagon secretion at low glucose concentrations (Fig. 1C and D). In both islets and {alpha}TC1-9 cells, GABA (10 µmol/l) significantly suppressed glucagon secretion to ~50% of control levels, whereas in the presence of SR95531 (10 µmol/l), exogenous GABA exerted no significant effect on glucagon secretion.

To quantify the likely contribution of endogenous GABA release to the control of glucagon secretion by glucose from isolated intact islets, we next examined the effects of the antagonist alone (Fig. 1E). At 0.5 mmol/l glucose, SR95531 had no effect on glucagon secretion, suggesting that endogenous GABA levels were low under these conditions. However, SR95531 significantly increased glucagon secretion to 125.8 ± 8.0 and 195.7 ± 25.2% of control levels at 5 and 10 mmol/l glucose, respectively (n = 7, P < 0.01), thus blunting the inhibitory effect of glucose at either concentration (Fig. 1E) but demonstrating a qualitatively more important role for GABA at 10 mmol/l glucose. Consistent with this selective GABAAR antagonist having no intrinsic or confounding nonspecific activity, incubation of clonal {alpha}TC1-9 cells with SR95531 in the absence of GABA exerted no effect on glucagon secretion stimulated by low glucose or at elevated glucose concentrations (Fig. 1F).

Expression of GABAARs in islets and clonal {alpha}-cells.
The above findings demonstrated the functional importance of GABAARs in suppressing glucagon secretion from mouse islets. To identify the expression pattern of GABAARs in mouse islets and {alpha}TC1-9 cells, we next used RT-PCR with subunit-specific primers (Fig. 2A). The detection of ß-actin was used as a positive control for all samples, whereas PCR was performed in the absence of the reverse transcription step as a negative control. The {alpha}4, ß3, and {gamma}2 and {varepsilon} subunits were expressed in both {alpha}TC1-9 cells and in islets, whereas mRNAs encoding the {alpha}3, ß2, and {delta} subunits were detected only in {alpha}TC1-9 cells. {alpha}5, {alpha}6, ß1, ß2, {gamma}1, and {gamma}3 subunits were absent from both. {alpha}1 mRNA was detected in mouse islets and {alpha}TC1-9 cells when RT-PCR was performed with previously published primer sequences (35,38) (data not shown), but sequencing revealed that this product was not related to Gabra1. Use of a novel primer set, specific for mouse Gabra1, produced a product of the expected size in brain, but not in islet cells (Fig. 2A), consistent with the absence of this subunit from the mouse endocrine pancreas.


Figure 2
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  		FIG. 2. A subset of GABAAR subunits is expressed in mouse pancreatic {alpha}-cells. RT-PCR was performed on total RNA from mouse brain, {alpha}TC1-9, or mouse islets using GABAAR subunit-specific primers (A). Positive (ß-actin, +) and negative controls (no RT), where reverse transcription was omitted, are shown. Immunocytochemistry with GABAAR subunit-specific antibodies demonstrated the presence of the GABAAR {alpha}4 and {gamma}2 subunits in {alpha}TC1-9 cells (B) and in isolated individual pancreatic islet {alpha}-cells identified by a pPPG-mRFP adenoviral construct (C). A control with no primary antibody added is shown (no 1°). Scale: 10 µm.

 
We confirmed the presence of GABAAR subunit proteins in mouse pancreatic {alpha}-cells using confocal immunocytochemistry. Clear cell surface expression of both {alpha}4 and {gamma}2 subunits was apparent in {alpha}TC1-9 cells (Fig. 2B). Individual {alpha}-cells were identified in dispersed mouse islets by infection with an adenovirus expressing mRFP under the control of the PPG promoter (Fig. 2C). This strategy is analogous to that recently used to record Ca2+ changes selectively in {alpha}-cells (5), where 92% (54/59 cells) of mRFP-labeled cells were also glucagon positive, while insulin-positive cells were uninfected (Fig. 2C, lower panels). Immunoreactivity against both {alpha}4 and {gamma}2 subunits was clearly evident at the cell surface of the mRFP-labeled primary {alpha}-cells (Fig. 2C). Thus, {alpha}TC1-9 cells and isolated mouse islets express a similar discrete subset of GABAAR subunits.

Glucose regulates the expression of GABAARs.
We next determined whether changes in glucose concentration might affect the expression of GABAAR subunits in intact isolated mouse islets. Culture for 24 h at increasing glucose concentrations (2.5, 5.5, and 16 mmol/l) strongly augmented the expression of GABAAR {alpha}4, ß3, and {gamma}2 mRNAs (Fig. 3A). Thus, after culture at 2.5 mmol/l glucose, expression of {alpha}4 subunit mRNA was 33.9 ± 13.3% of the level at 16 mmol/l (n = 3, P < 0.05), increasing to 73.4 ± 9.6% at 5.5 mmol/l glucose (n = 3, P < 0.05). Similar but less marked effects of glucose on ß3 and {gamma}2 mRNA expression were also observed (Fig. 3A).


Figure 3
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  		FIG. 3. Glucose dose-dependently increased the expression of GABAAR subunits in pancreatic {alpha}-cells. Quantitative real-time RT-PCR was used to assess the abundance of GABAAR {alpha}4, ß3, and {gamma}2 subunit mRNAs in islets cultured at 2.5, 5.5, and 16 mmol/l glucose (A) and in {alpha}TC1-9 cells cultured at 1, 3, 10, and 16 mmol/l glucose (B). Data are presented such that all treated levels are expressed relative to 16 mmol/l glucose control (= 1, dotted line) using the "{delta}-{delta} Ct" method (*P < 0.05, n = 3). A representative Western blot used for semiquantitative analysis (C) showing increasing {alpha}TC1-9 total protein (10, 20, and 40 µg) probed with a subunit-specific antibody for the {alpha}4 subunit. Relative subunit expression is expressed as the ratio of labeling intensity in 1.0 vs. 16 mmol/l glucose-treated cultures (D) (*P < 0.05, n = 4).

 
We next sought to determine the extent to which glucose affected GABAAR gene expression in {alpha}-cells, as opposed to other islet cell types. Because mouse {alpha}-cells cannot readily be purified (e.g., by fluorescence-activated cell sorting) (20), GABAAR expression was measured in clonal {alpha}TC1-9 cells. A similar dose-dependent increase in the expression of {alpha}4, ß3, and {gamma}2 mRNAs was evident in {alpha}TC1-9 cells cultured for 24 h at increasing glucose concentrations (1, 3, 10, and 16 mmol/l; Fig. 3B). Thus, after culture at 1 mmol/l glucose, expression of {alpha}4 subunit mRNA was 27.9 ± 2.4% of the level at 16 mmol/l (n = 3, P < 0.05), increasing to 52.1 ± 3.9% at 3 mmol/l glucose (n = 3, P < 0.05). As in the islets, the effects of glucose on ß3 and {gamma}2 mRNA expression were less marked, but each gene displayed significantly higher expression levels after culture at 16 mmol/l glucose compared with 1.0 mmol/l glucose. Correspondingly, Western (immuno)blot analysis revealed that GABAAR {alpha}4 subunit protein was significantly (~2.0-fold) higher after culture at 16 mmol/l compared with 1 mmol/l glucose (Fig. 3C).

Depolarizing concentrations of K+, but not insulin, regulate GABAAR expression.
The above data indicated that glucose is an important regulator of GABAAR expression in pancreatic {alpha}-cells. Because GABAAR expression has been shown to be modulated by depolarizing concentrations of K+ ions in neurons (41), we sought next to determine whether the effects of elevated glucose concentrations on GABAAR gene expression may be due, at least in part, to membrane hyperpolarization (42) or depolarization (3) and decreases in intracellular free [Ca2+]. Culture of {alpha}TC1-9 cells for 24 h at elevated (25 mmol/l) versus normal (5 mmol/l) K+ led to an inhibition of the expression of each isoform at 16 but not 1 mmol/l glucose, by 33.3 ± 3.7, 38.9 ± 5.9, and 36.8 ± 0.2% of control levels for {alpha}4, ß3, and {gamma}2 mRNA, respectively (Fig. 4A, n = 3, P < 0.05). By contrast, insulin (20 nmol/l) was without effect on the expression of any of the studied GABAAR isoforms in {alpha}TC1-9 cells (Fig. 4C), consistent with previous observations that insulin has no effect on GABAA receptor gene expression in neurons (43), while increasing the trafficking of the receptors to the cell surface both in these (43) and {alpha}-cells (23). In these experiments, we confirmed that the expression of each GABAAR mRNA was reduced in 1 mmol/l glucose, as in earlier experiments (Fig. 4B and D vs. Fig. 3).


Figure 4
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  		FIG. 4. Depolarizing concentrations of K+, but not insulin, regulate the expression of GABAAR subunits in {alpha}TC1-9 cells. Quantitative real-time RT-PCR was used to assess the abundance of GABAAR {alpha}4, ß3, and {gamma}2 subunit mRNAs in {alpha}TC1-9 cells cultured at 16 and 1 mmol/l glucose in the presence of 25 mmol/l K+ (A) or 20 nmol/l insulin (C). Data are presented such that all treated levels are expressed relative to control (= 1, dotted line) (5 mmol/l K+ or 0 nmol/l insulin) using the "{delta}-{delta} Ct" method (n = 3). In parallel experiments, we confirmed that at 5 mmol/l K+ (B) and at 0 nmol/l insulin (D), the abundance of each GABAAR subunit was reduced at 1 mmol/l glucose compared with 16 mmol/l glucose (= 1, dotted line). *P < 0.05.

 
We next examined whether the inhibitory effects of K+ depolarization or low glucose on GABAAR gene expression might be mediated by enhanced Ca2+ entry (Fig. 5), given the presence in {alpha}-cells of voltage-gated calcium channels (L-type and T-type) (3). Nimodipine (25 µmol/l), an L-type calcium-channel blocker that inhibits Ca2+ oscillations in {alpha}TC1-9 cells at low glucose concentrations (M.A.R., G.A.R., unpublished data), was without effect on GABAAR expression at 16 mmol/l glucose (Fig. 5) but abolished the inhibitory effects of low (1 mmol/l) glucose or high [K+]. Thus, diminished Ca2+ influx was necessary and sufficient for the enhancement of GABAAR {alpha}4, ß3, and {gamma}2 subunit expression at elevated glucose concentrations.


Figure 5
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  		FIG. 5. Effect of nimodipine on GABAAR expression. Quantitative real-time RT-PCR was used to assess the abundance of GABAAR {alpha}4, ß3, and {gamma}2 subunit mRNAs in {alpha}TC1-9 cells cultured at 16 or 1 mmol/l glucose in the absence or presence of 25 µmol/l nimodipine with 5 mmol/l K+ or nimodipine with 25 mmol/l K+. Data are presented such that all treated levels are expressed relative to control (= 1, dotted line) (16 mmol/l glucose with 5 mmol/l K+) using the "{delta}-{delta} Ct" method (*P < 0.05, n = 3).

 
To further investigate the possible mechanisms underlying the glucose-induced regulation of GABAAR expression, we examined the effects of the calcineurin (Ca2+/calmodulin-dependent protein phosphatase 2B) inhibitor cyclosporin A and the adenylate cyclase activator forskolin. In {alpha}TC1-9 cells, cyclosporin A (1 µmol/l) significantly increased the expression of ß3, but not {alpha}4, mRNA at low glucose concentrations (Fig. 6). Elevating cAMP levels by the addition of forskolin (10 µmol/l) significantly increased the expression of ß3 mRNA at both glucose concentrations tested, whereas {alpha}4 mRNA was only increased at 1 mmol/l glucose (Fig. 6).


Figure 6
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  		FIG. 6. Effects of cyclosporin A and forskolin on GABAAR expression. Quantitative real-time RT-PCR was used to assess the abundance of GABAAR {alpha}4 and ß3 subunit mRNAs in {alpha}TC1-9 cells cultured at 16 or 1 mmol/l glucose in the absence (control) or presence of either cyclosporin A (1 µmol/l) or forskolin (10 µmol/l). Data are presented such that all levels are expressed relative to control (16 mmol/l glucose with 5 mmol/l K+) (= 1, dotted line) using the "{delta}-{delta} Ct" method (*P < 0.05, n = 3).

 

   DISCUSSION
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Contribution of GABA to the regulation of glucagon secretion from mouse islets.
The first aim of this study was to reexamine the potential contribution of GABA release to the acute regulation of glucagon secretion from mouse islets. We have recently demonstrated that glucose can act directly on the mouse {alpha}-cell to inhibit Ca2+ oscillations (5), an effect that may be prompted by increased intracellular free ATP concentrations and closure of ATP-sensitive K+ channels—the latter causing cell depolarization and the inactivation of Na+-dependent action potentials (3). Although this study suggested that the release of insulin or Zn2+ was unlikely to be involved, a potential role for other paracrine mechanisms was not excluded.

Here, we show that release of endogenous GABA from neighboring ß-cells is likely to play a role in regulating glucagon secretion from mouse islets. Thus, exogenous GABA inhibited glucagon secretion at low glucose concentrations by 50–60% in both pancreatic mouse islets and murine {alpha}TC1-9 cells (Fig. 1A and B). These effects of GABA are thus substantially more than the 18% inhibition of secretion observed at 0 mmol/l glucose by Gilon et al. (20), a difference that may reflect either the use of alternative mouse strains (NMRI mouse in the earlier study) as well as differences in preculture (1 day in the present study) and incubation conditions (0 mmol/l glucose [20] vs. 0.5 mmol/l [present study]). From inspection of Fig. 1E, it is apparent that GABA release may contribute ~15 or ~40% toward the overall inhibitory effect of 5 or 10 mmol/l glucose on glucagon release, respectively. The greater apparent contribution of GABA release at higher glucose concentrations may reflect both 1) more substantial release of this neurotransmitter because ß-cells are progressively depolarized and 2) the "unmasking," in the absence of GABAAR function, of a stimulatory effect of high glucose concentration on glucagon secretion (7,44). Nonetheless, these contributions appear to be less than those estimated using the same approach in rat islets (22). Other observations point to differences in the relative importance of GABAergic regulation of glucagon release in mouse and rat. First, in the rat, blockade of GABAARs with SR95531 stimulated glucagon release both at low (1 mmol/l) and elevated (20 mmol/l) glucose concentrations, implying a tonic restraint by endogenous intra-islet GABA even under conditions where release of the neurotransmitter from ß-cells is unlikely to be stimulated (15). Second, SR95531 completely eliminated the inhibitory effect of elevated (20 mmol/l) glucose concentrations on glucagon secretion from rat islets (22), implying that enhanced GABA release might be the sole mechanism through which glucose acts to regulate glucagon secretion in this species. In contrast, the present findings in mouse are consistent with the view that the previously described metabolic effects of glucose on isolated mouse {alpha}-cells (5) are likely to play a significant role in the control of glucagon secretion in this species.

Expression of islet GABAARs in islets and regulation of gene expression by glucose.
Our results suggest that, in mouse pancreatic {alpha}-cells, the most abundant GABAAR complexes likely comprise 2{alpha}4, 2ß3, and {gamma}2/{varepsilon} subunits. Importantly, {alpha}4-containing GABAARs display a distinct pharmacology compared with complexes with other {alpha}-subunits, with high affinity for GABA (45), slow desensitization kinetics (46), and insensitivity to benzodiazepines (47). The putative paracrine regulator of glucagon secretion, Zn2+, co-released from ß-cells with insulin, might thus act at {alpha}-cell GABAARs. Zn2+ ions inhibit GABAAR function by an allosteric mechanism that depends on the subunit composition: {alpha}-ß subunit combinations show the highest sensitivity, although the incorporation of a {gamma}2 subunit reduces the inhibitory potency (48), as demonstrated in cell lines expressing human {alpha}4, ß3, and {gamma}2 GABAARs (45). In mouse islets, Zn2+ ions have no effect on glucagon secretion (5), but Zn2+ is reported to inhibit glucagon release from rat islets (9), possibly by opening {alpha}-cell KATP channels (3,10).

We report here for the first time that glucose regulates GABAAR gene expression in islets and implicates decreases in intracellular free [Ca2+] as a likely mediator of the effects of the sugar. We propose that by inhibiting electrical activity and hence Ca2+ oscillations, glucose removes an inhibitory regulator (which may be Ca2+ itself) for GABAAR gene expression. High K+ thus mimics the effect of glucose lowering (Fig. 4A), whereas nimodipine reverses this effect (Fig. 5). Calcium influx through N-type Ca2+ channels has been shown to be important in controlling glucagon secretion in mouse islets, but the role of the L-type Ca2+ channel is less clear (3). However, we have found that, in {alpha}TC1-9 cells, nimodipine completely blocked the effects of low glucose on glucagon secretion, whereas the N-type Ca2+ channel inhibitor w-conotoxin was without effect (M.A.R., G.A.R., unpublished data), suggesting differing roles for these channels in clonal versus primary {alpha}-cells. Nonetheless, our observations leave open the possibility that, in islets, {alpha}-cell L- and N-type channels may be somewhat selectively coupled to transcriptional or secretory events, respectively.

The mechanisms regulating GABAAR gene expression have so far largely been confined to the study of cerebellar granule cell neurons where depolarizing concentrations of K+ (41), cAMP (49), and mitogen-activated protein kinase regulation (50) affect GABAAR abundance. For example, culturing rat cerebellar granule neurons at 25 mmol/l K+ reduced ß3, as well as {alpha}1, {alpha}6, and ß2 subunit mRNA levels (41). The importance of Ca2+ entry-dependent pathways in the control of gene expression is well documented. One such mechanism is the regulation of cAMP response element binding protein (CREB) dephosphorylation by the Ca2+/calmodulin-dependent protein phosphatase 2B/calcineurin (51). Consistent with a possible role for calcineurin activation in the effects of low glucose, the calcineurin inhibitor cyclosporine A partially reversed the inhibitory effects of 1 mmol/l glucose on the expression of the ß3 receptor subunit (Fig. 6). Likewise, elevation of cAMP levels with forskolin increased the expression of both the {alpha}4 and ß3 subunits, albeit at both low and high glucose concentrations (see below). Inhibition of calcineurin can prolong CREB phosphorylation and stimulate CRE-mediated gene expression (52), and downregulation of GABAAR function by a calcineurin-dependent dephosphorylation of subunits has been shown earlier (53). Because CREB binding has recently been demonstrated at the promoters of the GABAAR ß3 and {gamma}2 subunit genes (54), we speculate that calcineurin-mediated dephosphorylation of CREB may suppress GABAAR gene expression in {alpha}-cells. However, the upregulation of a transcriptional repressor such as lost on transformation 1 (Lot1)/zinc finger regulator of apoptosis and cell cycle arrest (ZAC) when calcineurin is activated by low glucose (55) represents a further potential mechanism.

We have considered the possibility that an autocrine effect of glucagon release on GABAAR may be involved in the observed responses to glucose. Because, in neurons, elevated cAMP increased {alpha}1 and ß2, but decreased ß3 expression (49), loss of glucagon-stimulated increases in cAMP may potentially contribute to the increased expression of GABAAR ß3 and possibly {alpha}4 and ß3 subunits seen in {alpha}-cells at high glucose concentrations. Importantly, however, elevation of cAMP by forskolin did not overcome the decrease of GABAAR expression seen at low glucose concentrations (Fig. 6), indicating that other cAMP-independent mechanisms must also be involved in the effects of glucose.


   ACKNOWLEDGMENTS
 
This work was supported by grants to G.A.R. from the Wellcome Trust (Programme Grant 067081/Z/02/Z) and the Juvenile Diabetes Research Foundation (#1-2003-235). G.A.R. is a Wellcome Trust Research Leave Fellow.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received for publication May 24, 2006 and accepted in revised form November 2, 2006


   REFERENCES
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 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
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