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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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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<0.01 (Ctrl vs GABA), ## p<0.01 (GABA vs GABA+Bic or GABA+Ins).
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pone-0026225-g006: 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<0.01 (Ctrl vs GABA), ## p<0.01 (GABA vs GABA+Bic or GABA+Ins).

Mentions: We next conducted C-peptide radioimmunoassays (RIA) to determine whether or not the effect of insulin on the modulation of GABA-GABAAR system has an impact on GABAAR-mediated secretory function in INS-1 cells. As shown, consistent with our previous findings [15], GABA (30 µM) significantly increased C-peptide secretion in INS-1 cells, which was diminished by the GABAAR antagonist picrotoxin (50 µM) (Figure 6, p<0.01, n = 3), suggesting that GABA-induced insulin secretion in the β-cells is mediated by GABAAR. Furthermore, pre-treatment of the INS-1 cells with insulin (1 µM) resulted in a statistically-significant decrease in GABA-induced C-peptide secretion (Figure 6, p<0.01, n = 3). These results suggest that insulin-mediated inhibition of IGABA is related to down-regulation in GABA-induced insulin secretion 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)

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<0.01 (Ctrl vs GABA), ## p<0.01 (GABA vs GABA+Bic or GABA+Ins).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198728&req=5

pone-0026225-g006: 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<0.01 (Ctrl vs GABA), ## p<0.01 (GABA vs GABA+Bic or GABA+Ins).
Mentions: We next conducted C-peptide radioimmunoassays (RIA) to determine whether or not the effect of insulin on the modulation of GABA-GABAAR system has an impact on GABAAR-mediated secretory function in INS-1 cells. As shown, consistent with our previous findings [15], GABA (30 µM) significantly increased C-peptide secretion in INS-1 cells, which was diminished by the GABAAR antagonist picrotoxin (50 µM) (Figure 6, p<0.01, n = 3), suggesting that GABA-induced insulin secretion in the β-cells is mediated by GABAAR. Furthermore, pre-treatment of the INS-1 cells with insulin (1 µM) resulted in a statistically-significant decrease in GABA-induced C-peptide secretion (Figure 6, p<0.01, n = 3). These results suggest that insulin-mediated inhibition of IGABA is related to down-regulation in GABA-induced insulin secretion 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