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BAG3 regulates formation of the SNARE complex and insulin secretion.

Iorio V, Festa M, Rosati A, Hahne M, Tiberti C, Capunzo M, De Laurenzi V, Turco MC - Cell Death Dis (2015)

Bottom Line: This transiently disrupts the F-actin barrier and allows the redistribution of the insulin-containing granules to more peripheral regions of the β cell, hence facilitating insulin secretion.In this manuscript, we show for the first time that BAG3 plays an important role in this process.Moreover, we show that BAG3 binds to SNAP-25 and syntaxin-1, two components of the t-SNARE complex preventing the interaction between SNAP-25 and syntaxin-1.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacy, University of Salerno, via Giovanni Paolo II, 132, Fisciano, SA, Italy.

ABSTRACT
Insulin release in response to glucose stimulation requires exocytosis of insulin-containing granules. Glucose stimulation of beta cells leads to focal adhesion kinase (FAK) phosphorylation, which acts on the Rho family proteins (Rho, Rac and Cdc42) that direct F-actin remodeling. This process requires docking and fusion of secretory vesicles to the release sites at the plasma membrane and is a complex mechanism that is mediated by SNAREs. This transiently disrupts the F-actin barrier and allows the redistribution of the insulin-containing granules to more peripheral regions of the β cell, hence facilitating insulin secretion. In this manuscript, we show for the first time that BAG3 plays an important role in this process. We show that BAG3 downregulation results in increased insulin secretion in response to glucose stimulation and in disruption of the F-actin network. Moreover, we show that BAG3 binds to SNAP-25 and syntaxin-1, two components of the t-SNARE complex preventing the interaction between SNAP-25 and syntaxin-1. Upon glucose stimulation BAG3 is phosphorylated by FAK and dissociates from SNAP-25 allowing the formation of the SNARE complex, destabilization of the F-actin network and insulin release.

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BAG3 downregulation decreases insulin content levels and potentiates glucose-stimulated insulin secretion. (a) β-TC-6 cells at 80% confluence were transfected with a bag3-specific small interfering (si) RNA or a non-target (si) RNA (NT siRNA). After 48 h, whole-cell extracts were obtained and analyzed by western blot with an anti-BAG3 polyclonal antibody, anti-insulin antibody or, as a loading control, an anti-GAPDH monoclonal antibody. Densitometric analysis of insulin expressed as insulin/GAPDH ratios is reported in the lower panel. (b) Insulin secretory response of β-TC-6 cells transfected with a bag3-specific siRNA or an NT siRNA. The insulin secreted amount was evaluated by ELISA test on β-TC-6 supernatants collected at 15, 30 and 60 min after glucose stimulation. Time 0 represents insulin levels after starvation before addition of glucose. Data are mean±S.E.M. (n=2). *P<0.05, **P<0.01 and ***P<0.001. (c) β-TC-6 cells were transfected with a bag3-specific siRNA or an NT siRNA; 48 h after transfection cells were stimulated for 15 min with 25-mM glucose, fixed and stained with TRITC-conjugated phalloidin. All pictures are fully representative of multiple images from two independent experiments (scale bars, 10 μm)
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fig2: BAG3 downregulation decreases insulin content levels and potentiates glucose-stimulated insulin secretion. (a) β-TC-6 cells at 80% confluence were transfected with a bag3-specific small interfering (si) RNA or a non-target (si) RNA (NT siRNA). After 48 h, whole-cell extracts were obtained and analyzed by western blot with an anti-BAG3 polyclonal antibody, anti-insulin antibody or, as a loading control, an anti-GAPDH monoclonal antibody. Densitometric analysis of insulin expressed as insulin/GAPDH ratios is reported in the lower panel. (b) Insulin secretory response of β-TC-6 cells transfected with a bag3-specific siRNA or an NT siRNA. The insulin secreted amount was evaluated by ELISA test on β-TC-6 supernatants collected at 15, 30 and 60 min after glucose stimulation. Time 0 represents insulin levels after starvation before addition of glucose. Data are mean±S.E.M. (n=2). *P<0.05, **P<0.01 and ***P<0.001. (c) β-TC-6 cells were transfected with a bag3-specific siRNA or an NT siRNA; 48 h after transfection cells were stimulated for 15 min with 25-mM glucose, fixed and stained with TRITC-conjugated phalloidin. All pictures are fully representative of multiple images from two independent experiments (scale bars, 10 μm)

Mentions: We then tested the possibility that BAG3 influenced insulin levels and secretion. As shown in Figure 2a silencing BAG3 in β-TC-6 cells results in a significant reduction of their insulin content. Moreover, evaluation of insulin secretion in the supernatant by ELISA, following stimulation with 25-mM glucose, shows that this is strongly affected by BAG3 silencing. Under these experimental conditions there are two secretion peeks, one after 15 min and the other after 60 min (Figure 2b).44 BAG3 silencing results in increase of both early and late secretion. Similar results were obtained using a different siRNA to silence BAG3 excluding off-target effects (Supplementary Figure 1A).


BAG3 regulates formation of the SNARE complex and insulin secretion.

Iorio V, Festa M, Rosati A, Hahne M, Tiberti C, Capunzo M, De Laurenzi V, Turco MC - Cell Death Dis (2015)

BAG3 downregulation decreases insulin content levels and potentiates glucose-stimulated insulin secretion. (a) β-TC-6 cells at 80% confluence were transfected with a bag3-specific small interfering (si) RNA or a non-target (si) RNA (NT siRNA). After 48 h, whole-cell extracts were obtained and analyzed by western blot with an anti-BAG3 polyclonal antibody, anti-insulin antibody or, as a loading control, an anti-GAPDH monoclonal antibody. Densitometric analysis of insulin expressed as insulin/GAPDH ratios is reported in the lower panel. (b) Insulin secretory response of β-TC-6 cells transfected with a bag3-specific siRNA or an NT siRNA. The insulin secreted amount was evaluated by ELISA test on β-TC-6 supernatants collected at 15, 30 and 60 min after glucose stimulation. Time 0 represents insulin levels after starvation before addition of glucose. Data are mean±S.E.M. (n=2). *P<0.05, **P<0.01 and ***P<0.001. (c) β-TC-6 cells were transfected with a bag3-specific siRNA or an NT siRNA; 48 h after transfection cells were stimulated for 15 min with 25-mM glucose, fixed and stained with TRITC-conjugated phalloidin. All pictures are fully representative of multiple images from two independent experiments (scale bars, 10 μm)
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4385931&req=5

fig2: BAG3 downregulation decreases insulin content levels and potentiates glucose-stimulated insulin secretion. (a) β-TC-6 cells at 80% confluence were transfected with a bag3-specific small interfering (si) RNA or a non-target (si) RNA (NT siRNA). After 48 h, whole-cell extracts were obtained and analyzed by western blot with an anti-BAG3 polyclonal antibody, anti-insulin antibody or, as a loading control, an anti-GAPDH monoclonal antibody. Densitometric analysis of insulin expressed as insulin/GAPDH ratios is reported in the lower panel. (b) Insulin secretory response of β-TC-6 cells transfected with a bag3-specific siRNA or an NT siRNA. The insulin secreted amount was evaluated by ELISA test on β-TC-6 supernatants collected at 15, 30 and 60 min after glucose stimulation. Time 0 represents insulin levels after starvation before addition of glucose. Data are mean±S.E.M. (n=2). *P<0.05, **P<0.01 and ***P<0.001. (c) β-TC-6 cells were transfected with a bag3-specific siRNA or an NT siRNA; 48 h after transfection cells were stimulated for 15 min with 25-mM glucose, fixed and stained with TRITC-conjugated phalloidin. All pictures are fully representative of multiple images from two independent experiments (scale bars, 10 μm)
Mentions: We then tested the possibility that BAG3 influenced insulin levels and secretion. As shown in Figure 2a silencing BAG3 in β-TC-6 cells results in a significant reduction of their insulin content. Moreover, evaluation of insulin secretion in the supernatant by ELISA, following stimulation with 25-mM glucose, shows that this is strongly affected by BAG3 silencing. Under these experimental conditions there are two secretion peeks, one after 15 min and the other after 60 min (Figure 2b).44 BAG3 silencing results in increase of both early and late secretion. Similar results were obtained using a different siRNA to silence BAG3 excluding off-target effects (Supplementary Figure 1A).

Bottom Line: This transiently disrupts the F-actin barrier and allows the redistribution of the insulin-containing granules to more peripheral regions of the β cell, hence facilitating insulin secretion.In this manuscript, we show for the first time that BAG3 plays an important role in this process.Moreover, we show that BAG3 binds to SNAP-25 and syntaxin-1, two components of the t-SNARE complex preventing the interaction between SNAP-25 and syntaxin-1.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacy, University of Salerno, via Giovanni Paolo II, 132, Fisciano, SA, Italy.

ABSTRACT
Insulin release in response to glucose stimulation requires exocytosis of insulin-containing granules. Glucose stimulation of beta cells leads to focal adhesion kinase (FAK) phosphorylation, which acts on the Rho family proteins (Rho, Rac and Cdc42) that direct F-actin remodeling. This process requires docking and fusion of secretory vesicles to the release sites at the plasma membrane and is a complex mechanism that is mediated by SNAREs. This transiently disrupts the F-actin barrier and allows the redistribution of the insulin-containing granules to more peripheral regions of the β cell, hence facilitating insulin secretion. In this manuscript, we show for the first time that BAG3 plays an important role in this process. We show that BAG3 downregulation results in increased insulin secretion in response to glucose stimulation and in disruption of the F-actin network. Moreover, we show that BAG3 binds to SNAP-25 and syntaxin-1, two components of the t-SNARE complex preventing the interaction between SNAP-25 and syntaxin-1. Upon glucose stimulation BAG3 is phosphorylated by FAK and dissociates from SNAP-25 allowing the formation of the SNARE complex, destabilization of the F-actin network and insulin release.

Show MeSH
Related in: MedlinePlus