GABA Coordinates with Insulin in Regulating Secretory Function in Pancreatic INS-1 β-Cells

Posted on April 2, 2012

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Abstract

Pancreatic islet β-cells produce large amounts of γ-aminobutyric acid (GABA), which is co-released with insulin. GABA inhibits glucagon secretion by hyperpolarizing α-cells via type-A GABA receptors (GABAARs). We and others recently reported that islet β-cells also express GABAARs and that activation of GABAARs increases insulin release. Here we investigate the effects of insulin on the GABA-GABAAR system in the pancreatic INS-1 cells using perforated-patch recording. The results showed that GABA produces a rapid inward current and depolarizes INS-1 cells. However, pre-treatment of the cell with regular insulin (1 µM) suppressed the GABA-induced current (IGABA) by 43%. Zinc-free insulin also suppressed IGABA to the same extent of inhibition by regular insulin. The inhibition of IGABA occurs within 30 seconds after application of insulin. The insulin-induced inhibition of IGABA persisted in the presence of PI3-kinase inhibitor, but was abolished upon inhibition of ERK, indicating that insulin suppresses GABAARs through a mechanism that involves ERK activation. Radioimmunoassay revealed that the secretion of C-peptide was enhanced by GABA, which was blocked by pre-incubating the cells with picrotoxin (50 µM, p<0.01) and insulin (1 µM, p<0.01), respectively. Together, these data suggest that autocrine GABA, via activation of GABAARs, depolarizes the pancreatic β-cells and enhances insulin secretion. On the other hand, insulin down-regulates GABA-GABAAR signaling presenting a feedback mechanism for fine-tuning β-cell secretion.

Introduction

Gamma-aminobutyric acid (GABA) is a major neurotransmitter in the central nervous system (CNS), where GABA produces fast inhibition in mature neurons primarily by activation of A-type GABA receptor (GABAAR), a hetero-pentameric Cl channel [1]. A large amount of GABA is also produced in the pancreatic islet [2], where it exists at the highest concentration outside of the CNS [3]. Pancreatic GABA is primarily produced by the β-cell [4], in which GABA is stored in synaptic-like microvesicles that are distinct from insulin-containing large-dense core vesicles (LDCVs) [5]. However, recent evidence indicates that GABA is co-localized with insulin in LDCVs in human islets and that the release of GABA from the β-cells is glucose-dependent [6]. The release of GABA from β-cells is “tonic” [7], [8], yet the amount of released GABA is regulated by the metabolic state of β-cells [9].

In the pancreatic islet, GABA released from β-cells plays a critical role in the regulation of glucagon secretion from α-cells. Specifically, GABA activates GABAARs in α-cells, sequentially leading to an influx of Cl and membrane hyperpolarization, and hence an inhibition of glucagon secretion. The GABAAR-mediated hyperpolarization of α-cells represents a physiological mechanism for glucose-induced suppression of glucagon release because blockade of GABAAR diminishes the inhibitory effect of high glucose on glucagon secretion in isolated rat [10] or mouse [11] islets. In relation to this notion, we have recently demonstrated that insulin suppresses glucagon secretion by enhancing intra-islet GABA-GABAAR signaling through translocation of GABAAR from an intracellular pool to the cell surface of α-cells [12].

Studies, including ours, have demonstrated that GABAARs are also expressed in the primary islet β-cells [12], [13] and insulin-secreting clonal β-cell lines [14], [15]. Unlike in mature neurons and α-cells, stimulation of GABAARs in β-cells induces membrane depolarization, enhancing insulin secretion in the presence of physiological concentrations of glucose [6], [15]. Consistent with the notion that the autocrine insulin is essential for β-cell function [16], [17], we recently demonstrated that GABA, in cooperation with insulin, enhances the proliferation and survival of the β-cells through activation of the PI3-K/Akt pathway. Remarkably, GABA promotes β-cell regeneration and reverses diabetes in mouse models [18]. In the present study, we found that insulin negatively regulates GABAAR function and inhibits GABA-induced β-cell secretion. Our results demonstrated a feedback mechanism that fine-tunes β-cell secretion.

Discussion

Pancreatic β-cells produce a large amount of GABA [27], whereas GABAARs are expressed in both β-cells [6], [15] and α-cells [7], [12]. In α-cells, GABA hyperpolarizes the membrane potential and suppresses glucagon secretion [7], [12], via a mechanism involving PI3-K/Akt signaling dependent GABAAR plasma membrane translocation [12]. In contrast, we and others demonstrated that GABA depolarizes β-cells and stimulates insulin secretion from these cells [6], [15]. These observations suggest that GABA, as a paracrine or autocrine factor plays an important role within pancreatic islets in the regulation of islet cell secretion and function. In the present study, we sought to investigate how insulin affects GABA-GABAAR system in the β-cells and thereby modulates its secretory pathways.

In INS-1 cells, glucose induces a gradual and sustained depolarization, whereas GABA produces rapid and bicuculline- or picrotoxin-sensitive membrane depolarization, associated with remarkable increases in intracellular Ca2+ concentration and insulin secretion. A recent study by Braun et al. suggested that glucose stimulates feed-forward release of GABA from the β-cells [6]. Furthermore, the GABA-stimulated insulin release appears to be glucose concentration-dependent [6], [15]. Of note, our results showed that GABAAR antagonist picrotoxin attenuated about 50% of the GABA-induced C-peptide release. This is likely due to the fact that GABA-stimulated insulin secretion in the β-cells is partially contributed by activation of B-type GABA receptor (GABABR) [28]. These observations suggest that the autocrine GABA-GABAAR system in β-cells constitutes an effective signaling component of the glucose-sensing machinery.

The opposite effects of GABA in the two types of islet endocrine cells are likely because β-cells and α-cells have different Cl reversal potential (ECl). The direction of Cl flow upon opening of the GABAAR channel is dependent on the electrochemical driving force which is determined by the resting membrane potential and the ECl [29]. For example, in the early developing brain, GABA induces depolarizing effects in immature neurons [30], while it exerts inhibitory effects by hyperpolarizing the membrane potential in mature neurons of the adult brain [31]. The switch from excitation to inhibition of GABAAR activation is due to a shift of ECl which is controlled by increased activity of K+-Cl co-transporter-2 (KCC2) in the brain during development [32]. In this regard, functional KCC has been identified in pancreatic α-cells, but not in the β-cells [33], [34].

Regular human insulin is a complex of insulin and zinc [35]. The finding that zinc-free insulin suppressed IGABA to a degree similar to that of regular insulin suggests that the inhibitory effects of insulin on IGABA is dependent on the insulin peptide. It is interesting to note that application of zinc-free insulin together with GABA did not inhibit IGABA, whereas pre-treating the cell for at least 30 seconds with zinc-free insulin inhibited IGABA. These results suggest that insulin-induced inhibition of IGABA in INS-1 cells requires insulin signaling processes. Such inhibitory effects of insulin on GABA-induced current was also observed in the non-islet β-cells [36] The potentiating effect of insulin on IGABA in neurons and α-cells is attributed to GABAAR insertion into the plasma membrane, which occurred about 10-15 min after insulin treatment [12], [25]. Under similar experimental conditions, however, we did not observe increased GABAAR localization at the plasma membrane upon insulin treatment in INS-1 cells. Furthermore, unlike in the α-cells, where the insulin-enhanced IGABA is PI3-K/Akt dependent, our data does not suggest the involvement of PI3-K/Akt signaling in the inhibition of IGABA by insulin in the β-cells. In contrast, MEK/ERK inhibitor PD98059 blocked the inhibitory effect of insulin on GABA-induced current, suggesting that insulin regulates GABAAR function in INS-1 cells via activation of the MEK/ERK signaling pathway.

The explanation for the opposite effects of insulin on GABAAR in α- and β-cells is largely unknown, although it may be due to the different subunit composition of GABAAR in the two types of islet cells [12], [15]. It is interesting to note that in neurons, activation of the insulin-PI3-K signaling pathway enhances IGABA due to the increase in cell surface-localized GABAAR, whereas activation of insulin receptor with ERK kinase causes inhibition of IGABA through phosphorylation of a specific subunit of GABAAR [36], [37]. Particularly, α-subunits of the GABAAR have a putative phosphorylation site for ERK [37]. Presumably, such phosphorylation occurs on an intracellular site allowing immediate allosteric modifications of GABAAR.

Given the relatively rapid inhibitory effect of insulin on IGABA, it is possible that insulin may also act as a non-competitive inhibitor of the GABAAR in the β-cells, as has been observed in non-β-cells [36]. In relation to this notion, it has been reported that a direct receptor-receptor interaction occurs between GABAAR and dopamine D5 receptor, which affects the GABAAR activation [38]. Further study is warranted to test if there is an interaction between insulin receptor and GABAAR, and to determine the molecular mechanism by which insulin modulates GABA-GABAAR signaling in the β-cells.

In INS-1 cells, insulin suppresses IGABA and decreases GABA-mediated insulin secretion in the β-cells which suggests that insulin may utilize the GABA-GABAAR system to constitute a feedback mechanism for the β-cell secretion. Our findings are in a good agreement with previous observations suggesting that activation of insulin receptor inhibits insulin secretion in the β-cells [39]. Conversely, inhibition of PI3-K signaling pathways enhances insulin secretion in the β-cells [40], [41]. A study by Khan et al suggested that insulin inhibits insulin secretion through activation of KATP channels in the β-cells [42]. A study by Jimenez-Feltstrom and colleagues [43] suggested that the effect of insulin on IGABA is insulin-dose dependent, exemplified by the observation that, insulin, at low concentrations (i.e., from 0.05 to 0.1 nM) stimulated insulin release, while at concentrations higher than 250 nM, insulin inhibited insulin secretion from the β-cells.

It should be noted that under certain circumstance, effects of insulin on IGABA are excitatory [16]. These previous reports that describe the stimulatory effects of insulin on β-cell secretory process were mostly supported by experiments involving β-cells from organisms with genetic knockout or overexpression of the insulin receptor [44][48], The different outcomes imply that the modulation of insulin on the GABA-GABAAR system in the β-cells may be dependent on their metabolic status.

The physiological relevance of GABA signaling in the regulation of islet β-cell function has yet to be fully identified. We demonstrated recently that the depolarizing effects of GABA may lead to activation of PI3-K/Akt dependent cell growth and survival pathways in the β-cells [18]. Insulin is an important positive autocrine regulator of β-cell growth and survival [17], [49]. GABA, when co-released with insulin [6], synergistically enhances insulin-stimulated cell growth and survival pathways in the β-cells [18]. In support of previous findings that insulin is a negative regulator of insulin secretion [39][42], [50], our data suggest that insulin utilizes the autocrine GABA-GABAAR pathway to operate its negative feedback suppression in the β-cells.

Such a negative feedback modulator appears to be important for maintaining islet hormones at appropriate levels [51]. It is conceivable that basal insulin may serve as a maintenance signal that primes the β-cell to respond to subsequent glucose stimulus, insulin may utilize GABA-GABAAR system to inhibit further release at the peak of the exocytotic event, particularly, at very high local insulin concentration. Previous euglycemic hyperinsulinemic clamp studies in humans suggest that this negative short-loop insulin-β-cell feedback is an important mechanism in maintaining appropriate β-cell secretion, since inadequate feedback suppression is found in obese patients, and may partly account for their prevailing hyperinsulinemia [52]. Given that autocrine insulin action is critical in maintaining normal β-cell function [16], [17], and that β-cell insulin resistance can deteriorate β-cell function that accelerates the progression of diabetes [53], [54], future studies are required to determine whether the impairment of the autocrine insulin-GABA-GABAAR signaling contributes to β-cell insulin resistance in type 2 diabetes.

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