Correlation between GABA release from rat islet beta -cells and their metabolic state

Frederic Winnock, Zhidong Ling, Rene De Proft, Sandra Dejonghe, Frans Schuit, Frans Gorus, and Daniel Pipeleers

Diabetes Research Center, Vrije Universiteit Brussel, B-1090 Brussels, Belgium


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Pancreatic beta -cells express glutamate decarboxylase (GAD), which is responsible for the production and release of gamma -aminobutyric acid (GABA). Over a 24-h culture period, total GABA release by purified rat beta -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 beta -cell activities were inhibited by addition of diazoxide, verapamil, or cycloheximide or by reduction of extracellular calcium levels; it was counteracted when beta -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 beta -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 beta -cells, and hence GABA production and release, is compatible with previous findings on ATP suppression of brain GAD activity.

diabetes; glutamate decarboxylase; insulin


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IDENTIFICATION OF ISLET CELL ANTIBODIES that recognize glutamate decarboxylase (GAD) in the pancreatic beta -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 beta -cells under physiological conditions. Pancreatic beta -cells rank among the few nonneuronal cell types that express GAD and contain its product, gamma -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 beta -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 beta -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 beta -cell functions.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation and culture of rat islets and purified beta -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 beta -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 beta -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 beta -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 of beta -cell extracts (18). Anti-GAD 65 antibody was purchased from Chemicon (Chemicon International, Temecula, CA) and used at 1:1,000 dilution. The intensity of the bands was quantified by National Institute of Health Image 1.61 software and expressed in arbitrary units of optical density.

Statistical 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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Comparison of GABA release from isolated islets and purified beta -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 beta -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 beta -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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Table 1.   Effects of glucose on GABA release by isolated islets and from purified beta -cells



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Fig. 1.   Effect of glucose metabolism on GABA release by beta -cells. Data are means ± SE of 4 independent experiments. Statistical significance (* P < 0.05) of differences induced by dinitrophenol (DNP) or mannoheptulose (MHP) are calculated by paired Student's t-test vs. the 6 or 20 mM glucose control.

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 beta -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).

On the other hand, this glucose-induced suppression was amplified in the presence of the translation inhibitor cycloheximide or the secretory inhibitors diazoxide and verapamil (Fig. 2). The experiments with cycloheximide were conducted at 0.3 mM calcium to reduce the secretory activity. As expected, these conditions inhibited glucose-induced insulin release (Fig. 2).


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Fig. 2.   GABA release during suppression of synthetic or secreting activities. Data represent means ± SE of 3-5 independent experiments. Statistical significance (* P < 0.05) of differences induced by cycloheximide (CHX), diazoxide (DIAZ), or verapamil (VER) was calculated by paired Student's t-test vs. the 3, 6, 10, or 20 mM control condition.

Effect of nonmetabolized stimuli of beta -cell functions. Glibenclamide, glucagon, and GLP-1 are nonmetabolized stimuli of beta -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 beta -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 beta -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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Fig. 3.   Role of calcium and cAMP on glucose effect. Data are means ± SE of 3-4 independent experiments. Statistical significance [* P < 0.05 or lack of significance (NS)] of differences induced by glibenclamide (Glib), glucagon (Gluca), glucagon-like peptide-1 (GLP-1), IBMX, and IBMX+RP isomer of 8-BrcAMP (PKI) was calculated by paired Student's t-test vs. corresponding control condition.

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 beta -cells that were cultured for 24 h at low (3 mM) or high (20 mM) glucose (Fig. 4); GAD65 expression was comparable at 0.25 or 2 mM glutamine, indicating that it was not influenced by GABA production or release during these experimental conditions (Fig. 4). No statistically significant differences were measured in GAD activity of cell extracts after culture at low or high glucose, irrespective of the presence of PLP (Fig. 5).


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Fig. 4.   Effect of glucose on glutamate decarboxylase (GAD)65 expression in Western blots. Immunoblot of GAD65 in beta -cells after 24-h culture at 3 or 20 mM glucose with 0.25 or 2 mM glutamine is shown. This figure is representative of 4 independent experiments; there was no statistically significant difference in optical density units in the 4 lanes: 3.3 ± 1.1, 3.3 ± 0.5, 3.9 ± 0.8, and 3.5 ± 0.7 for, respectively, 3 mM glucose (0.25 mM glutamine, 2 mM glutamine) and 20 mM glucose (0.25 mM glutamine, 2 mM glutamine).



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Fig. 5.   Effect of glucose on GAD activity in cell extracts. GAD activity was measured in beta -cell extracts incubated for 60 min in the absence or presence of pyridoxal 5'-phosphate (PLP). Data represent means ± SE of 3 independent experiments. Statistical significance (* P < 0.05) of difference from control was calculated by Student's t-test.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study confirms that the amount of GABA release from rat beta -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 beta -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 beta -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 beta -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. beta -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 beta -cells. We compared the expression of the GAD65 protein and the GAD activity in extracts of beta -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. beta -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 beta -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 beta -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 beta -cells, which would be compatible with previous observations on brain GAD. Further investigations on local ATP levels in beta -cells should test this hypothesis.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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