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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-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.
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pone-0026225-g004: 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.

Mentions: PI3-K is a key signaling molecule that mediates the trophic effects of insulin [24]. We therefore examined whether insulin-induced inhibition of IGABA requires involvement of PI3-K. INS-1 cells were pretreated with 100 nM of the specific PI3-K inhibitor wortmannin for 10 minutes, and then treated with 1 µM zinc-free insulin prior to measurement of IGABA. As shown (Figures 4A and 4B), the zinc-free insulin-induced inhibition of IGABA persisted in the presence of PI3-K inhibitor, which is suggestive of a PI3-K independent process. To confirm this finding, we transfected INS-1 cells with a vector expressing a dominant-negative form of Akt (DN-Akt) and tagged with green fluorescent protein (GFP) [12]. The dominant negative effect of DN-Akt was determined in parallel experiments by Western Blot using anti-phospho-Akt (Ser473) antibody in either transfected or non-transfected INS-cells treated with or without inhibitors as indicated (Figure 4C). Application of zinc-free insulin (1 µM) to the transfected INS-1 cells still caused a remarkable reduction of IGABA (Figures 4D). Normalized IGABA from separated experiments showed zinc-free insulin reduced IGABA by approximately 30% in INS-1 cells expressing DN-Akt (Figure 4E, n = 5, p<0.05). These observations suggest that zinc-free insulin-induced inhibition on IGABA is not sensitive to the PI3-K/Akt inhibition.


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-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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026225-g004: 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.
Mentions: PI3-K is a key signaling molecule that mediates the trophic effects of insulin [24]. We therefore examined whether insulin-induced inhibition of IGABA requires involvement of PI3-K. INS-1 cells were pretreated with 100 nM of the specific PI3-K inhibitor wortmannin for 10 minutes, and then treated with 1 µM zinc-free insulin prior to measurement of IGABA. As shown (Figures 4A and 4B), the zinc-free insulin-induced inhibition of IGABA persisted in the presence of PI3-K inhibitor, which is suggestive of a PI3-K independent process. To confirm this finding, we transfected INS-1 cells with a vector expressing a dominant-negative form of Akt (DN-Akt) and tagged with green fluorescent protein (GFP) [12]. The dominant negative effect of DN-Akt was determined in parallel experiments by Western Blot using anti-phospho-Akt (Ser473) antibody in either transfected or non-transfected INS-cells treated with or without inhibitors as indicated (Figure 4C). Application of zinc-free insulin (1 µM) to the transfected INS-1 cells still caused a remarkable reduction of IGABA (Figures 4D). Normalized IGABA from separated experiments showed zinc-free insulin reduced IGABA by approximately 30% in INS-1 cells expressing DN-Akt (Figure 4E, n = 5, p<0.05). These observations suggest that zinc-free insulin-induced inhibition on IGABA is not sensitive to the PI3-K/Akt inhibition.

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