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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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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.
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pone-0026225-g003: 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.

Mentions: Clinically-used insulin contains zinc [21] and zinc inhibits IGABA in neurons [22] by directly binding to GABAAR channel and lowering its open probability [23]. Therefore, we used zinc-free insulin to verify the suppressive effects of insulin on IGABA. Our result showed that the zinc-free insulin also suppressed IGABA in INS-1 cells (Figure 3A, p<0.05). The efficacy of zinc-free insulin was similar to that of regular insulin (Figures 3B and 3C). However, the suppression of IGABA by insulin disappeared when zinc-free insulin was applied simultaneously with GABA (Figures 3D and 3E, p>0.05, n = 3). We found that the reduction of IGABA was seen only when zinc-free insulin was pre-applied to the cell (i.e., >30 seconds) prior to GABA application (Figures 3D and 3E, p<0.05, n = 3). These results suggest that zinc-free insulin-induced inhibition of IGABA is not a result of direct blockade of GABAAR channels, but rather through a signaling process.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026225-g003: 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.
Mentions: Clinically-used insulin contains zinc [21] and zinc inhibits IGABA in neurons [22] by directly binding to GABAAR channel and lowering its open probability [23]. Therefore, we used zinc-free insulin to verify the suppressive effects of insulin on IGABA. Our result showed that the zinc-free insulin also suppressed IGABA in INS-1 cells (Figure 3A, p<0.05). The efficacy of zinc-free insulin was similar to that of regular insulin (Figures 3B and 3C). However, the suppression of IGABA by insulin disappeared when zinc-free insulin was applied simultaneously with GABA (Figures 3D and 3E, p>0.05, n = 3). We found that the reduction of IGABA was seen only when zinc-free insulin was pre-applied to the cell (i.e., >30 seconds) prior to GABA application (Figures 3D and 3E, p<0.05, n = 3). These results suggest that zinc-free insulin-induced inhibition of IGABA is not a result of direct blockade of GABAAR channels, but rather through a signaling process.

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