GABA coordinates with insulin in regulating secretory function in pancreatic INS-1 β-cells

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 (GABA(A)Rs). We and others recently reported that islet β-cells also express GABA(A)Rs and that activation of GABA(A)Rs increases insulin release. Here we investigate the effects of insulin on the GABA-GABA(A)R 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 (I(GABA)) by 43%. Zinc-free insulin also suppressed I(GABA) to the same extent of inhibition by regular insulin. The inhibition of I(GABA) occurs within 30 seconds after application of insulin. The insulin-induced inhibition of I(GABA) persisted in the presence of PI3-kinase inhibitor, but was abolished upon inhibition of ERK, indicating that insulin suppresses GABA(A)Rs 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 GABA(A)Rs, depolarizes the pancreatic β-cells and enhances insulin secretion. On the other hand, insulin down-regulates GABA-GABA(A)R signaling presenting a feedback mechanism for fine-tuning β-cell secretion.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. GABA depolarizes membrane potential and increases intracellular Ca2+ in INS-1 cells.

(A) Perfusion of ECS containing 28 mM glucose induces a gradual and sustained depolarization of the membrane potential (Vm) (n = 5). (B) GABA induces a rapid and GABAAR inhibition-sensitive depolarization of Vm under the current-clamp conditions at 1.4 mM glucose (n = 5). (C) Cells cultured in 96-well plates pre-loaded with Fluo-3 AM were treated with GABA (30 µM), or 5 mM KCl as positive control. Changes in relative fluorescence units (RFU) were monitored with a fluorescent plate reader. Data are Mean±SE, n = 6.

Figure 2. GABA-evoked currents (IGABA) is inhibited by insulin in INS-1 cells.

GABA-evoked inward current was measured by means of a computer-controlled multi-barrelled perfusion system, in two-minute intervals, under voltage-clamp conditions. Representative traces of GABA-evoked currents in the absence and presence of insulin (100 nM, (A), 1 µM, (B)) in the same INS-1 cell. A' and B' represents the average of IGABA from separated experiments. (C) Normalized average IGABA during the course of experiment (control = average of first 4 IGABA, insulin = average of IGABA in the presence insulin at indicated concentrations). Data were mean ± SE. *p<0.05 ** p<0.01, n = 6.

Figure 3. Zinc-free insulin inhibits GABA-evoked currents in INS-1 cells.

(A) Representative traces of GABA-evoked currents in the absence and presence of zinc-free insulin and regular insulin in the same INS-1 cell. (B) The average of IGABA from separated experiments. (C) Normalized average IGABA was separately recorded during the course of experiment (control = average of first 4 IGABA, ZFI = average of IGABA in the presence zinc-free insulin, insulin = average of IGABA in the presence of insulin after washing out). (D) Representative traces of IGABA obtained from when GABA was applied simultaneously with insulin or 30 seconds after insulin pre-treatment. (E) Normalized average IGABA of separated experiments as described in (C). Data were mean ± SE. *p<0.05, n = 5.

Figure 4. Insulin-induced inhibition of IGABA in INS-1 cells is PI3-K/Akt independent.

(A) Representative traces of GABA-evoked currents in the absence and presence of zinc-free insulin (1 µM) along with PI3-K inhibitor wortmannin (100 nM). (B) Normalized average of IGABA from separated experiments. (C) Akt activity determined by Western Blotting using anti-phospho Akt (S473) in cells treated without or with wortmannin (Wort), or in the cells transfected with dominant-negative Akt (DN-Akt). (D) Representative traces of GABA-evoked currents in cells expressing DN-Akt in the absence and presence of zinc-free insulin (ZFI,1 µM). (E) Average IGABA from separated time-course experiments. Data were mean ± SE. *p<0.05, ** p<0.01, n = 5.

Figure 5. Insulin suppresses IGABA which is not associated with GABAAR membrane relocalization and is ERK-dependent.

(A) Confocal microscopic image of INS-1 cells immunostained for GABAARs using anti-GABAAR β2/3 mouse IgG and Cy3-conjugated secondary antibody (red) with DAPI-nuclear staining (blue). Cells were treated with or without insulin, in the presence or absence of PI3-K inhibitor wortmannin. (B) Insulin (100 nM, 5 min) stimulated ERK phosphorylation in INS-1 cells, which was blocked by pre-treatment of the cells with PD98059 (20 µM, 10 min). (C) Representative traces of GABA-evoked currents in the absence and presence of zinc-free insulin (0.6 µM) with or without PD98059 (20 µM). (D) Normalized average of IGABA from separated experiments. Data were mean ± SE. *p<0.05, ** p<0.01, n = 5–6.

Figure 6. GABA enhances insulin secretion which is attenuated by insulin in INS-1 cells.

Insulin secretion was evaluated by C-peptide RIA. Cells were serum-starved in KRB buffer containing 1.4 mM glucose for 60 min prior to the RIA. The RIA was conducted using cells which had their culture medium replaced with fresh KRB buffer containing 1.4 mM glucose (or 11.1 mM glucose as positive control, PC), in the presence of GABA (30 µM), with or without either picrotoxin (Pic, 50 µM) or insulin (Ins, 1 µM) for 120 min. Data were mean ± SE, from three independent experiments with each sample counted in triplication. ** p