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

Bansal P, Wang S, Liu S, Xiang YY, Lu WY, Wang Q - PLoS ONE (2011)

Bottom Line: 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.On the other hand, insulin down-regulates GABA-GABA(A)R signaling presenting a feedback mechanism for fine-tuning β-cell secretion.

View Article: PubMed Central - PubMed

Affiliation: Departments of Physiology and Medicine, University of Toronto, Toronto, Ontario, Canada.

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.

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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.
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pone-0026225-g005: 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.

Mentions: We previously demonstrated that insulin enhances the insertion of GABAAR into the plasma membrane in neuron [25] and α-cell [12]. We thus investigated whether insulin could alter GABAAR membrane expression in INS-1 cells by immunostaining using antibody against the GABAARβ2/3 subunits. As shown, insulin (1 µM, 15 min) did not alter the staining profile of GABAARβ2/3 subunits (Figure 5A) in INS-1 cells treated with or without wortmannin, suggesting that insulin-induced suppression of IGABA is not related to GABAAR redistribution in INS-1 cells.


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

Bansal P, Wang S, Liu S, Xiang YY, Lu WY, Wang Q - PLoS ONE (2011)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198728&req=5

pone-0026225-g005: 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.
Mentions: We previously demonstrated that insulin enhances the insertion of GABAAR into the plasma membrane in neuron [25] and α-cell [12]. We thus investigated whether insulin could alter GABAAR membrane expression in INS-1 cells by immunostaining using antibody against the GABAARβ2/3 subunits. As shown, insulin (1 µM, 15 min) did not alter the staining profile of GABAARβ2/3 subunits (Figure 5A) in INS-1 cells treated with or without wortmannin, suggesting that insulin-induced suppression of IGABA is not related to GABAAR redistribution in INS-1 cells.

Bottom Line: 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.On the other hand, insulin down-regulates GABA-GABA(A)R signaling presenting a feedback mechanism for fine-tuning β-cell secretion.

View Article: PubMed Central - PubMed

Affiliation: Departments of Physiology and Medicine, University of Toronto, Toronto, Ontario, Canada.

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.

Show MeSH
Related in: MedlinePlus