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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 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.
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pone-0026225-g001: 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.

Mentions: As previously demonstrated [20], under current-clamp conditions INS-1 cells displayed a quiescent membrane potential around −60 mV when ECS contained 1.4 mM glucose (Figure 1A). Perfusion of the cell with ECS containing 28 mM glucose caused a gradual and sustained depolarization. In some cases, bursts of action potentials were superimposed on the glucose-induced depolarization (Figure 1A). Under the same recording conditions, perfusion of GABA induced a fast membrane depolarization in the INS-1 cell (Figure 1B). The GABA-induced depolarization was completely blocked by GABAAR antagonist picrotoxin (Figure 1B) or largely attenuated by bicuculline (not shown), consistent with our previous findings, and those of others in the same cell line or isolated human islet beta β-cells [6], [18]. These results suggest that GABA, via activation of GABAAR, induces membrane potential depolarization in pancreatic 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 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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026225-g001: 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.
Mentions: As previously demonstrated [20], under current-clamp conditions INS-1 cells displayed a quiescent membrane potential around −60 mV when ECS contained 1.4 mM glucose (Figure 1A). Perfusion of the cell with ECS containing 28 mM glucose caused a gradual and sustained depolarization. In some cases, bursts of action potentials were superimposed on the glucose-induced depolarization (Figure 1A). Under the same recording conditions, perfusion of GABA induced a fast membrane depolarization in the INS-1 cell (Figure 1B). The GABA-induced depolarization was completely blocked by GABAAR antagonist picrotoxin (Figure 1B) or largely attenuated by bicuculline (not shown), consistent with our previous findings, and those of others in the same cell line or isolated human islet beta β-cells [6], [18]. These results suggest that GABA, via activation of GABAAR, induces membrane potential depolarization in pancreatic 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