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Published on: June 25, 2012
VAV2, a guanine nucleotide exchange factor for Rac1, regulates glucose-stimulated insulin secretion in pancreatic
Rajakrishnan Veluthakal1,2, Ragadeepthi Tunduguru3, Daleep Kumar Arora4
1Beta Cell Biochemistry Laboratory, John D. Dingell VA Medical Center, 4646 John R, Detroit, MI, 48201, USA.
Aims/Hypothesis:
Rho GTPases (Ras-related C3 botulinum toxin substrate 1 [Rac1] and cell division cycle 42 [Cdc42]) have been shown to regulate glucose-stimulated insulin secretion (GSIS) via cytoskeletal remodelling, trafficking and fusion of insulin-secretory granules with the plasma membrane. GTP loading of these G proteins, which is facilitated by GDP/GTP exchange factors, is a requisite step in the regulation of downstream effector proteins. Guanine nucleotide exchange factor VAV2 (VAV2), a member of the Dbl family of proteins, has been identified as one of the GDP/GTP exchange factors for Rac1. Despite recent evidence on the regulatory roles of VAV2 in different cell types, roles of this guanine nucleotide exchange factor in the signalling events leading to GSIS remain undefined. Using immunological, short interfering RNA (siRNA), pharmacological and microscopic approaches we investigated the role of VAV2 in GSIS from islet beta cells.
Methods:
Co-localisation of Rac1 and VAV2 was determined by Triton X-114 phase partition and confocal microscopy. Glucose-induced actin remodelling was quantified by live cell imaging using the LifeAct-GFP fluorescent biosensor. Rac1 activation was determined by G protein linked immunosorbent assay (G-LISA).
Results:
Western blotting indicated that VAV2 is expressed in INS-1 832/13 beta cells, normal rat islets and human islets. Vav2 siRNA markedly attenuated GSIS in INS-1 832/13 cells. Ehop-016, a newly discovered small molecule inhibitor of the VAV2-Rac1 interaction, or siRNA-mediated knockdown of VAV2 markedly attenuated glucose-induced Rac1 activation and GSIS in INS-1 832/13 cells. Pharmacological findings were recapitulated in primary rat islets. A high glucose concentration promoted co-localisation of Rac1 and VAV2. Real-time imaging in live cells indicated a significant inhibition of glucose-induced cortical actin remodelling by Ehop-016.
Conclusions/Interpretation:
Our data provide the first evidence to implicate VAV2 in glucose-induced Rac1 activation, actin remodelling and GSIS in pancreatic beta cells.
Insights
VAV2 is crucial for glucose-stimulated insulin secretion (GSIS) by regulating Rac1 activation and actin remodelling in pancreatic beta cells. This study identifies VAV2 as a key player in GSIS signaling pathways.
Area of Science:
- Cellular and Molecular Biology
- Endocrinology
- Signal Transduction
Background:
- Rho GTPases, including Rac1 and Cdc42, regulate insulin secretion through cytoskeletal dynamics and granule exocytosis.
- GDP/GTP exchange factors (GEFs) are essential for activating Rho GTPases, but the role of VAV2, a GEF for Rac1, in glucose-stimulated insulin secretion (GSIS) remains unclear.
Purpose of the Study:
- To investigate the role of VAV2 in regulating Rac1 activation, actin dynamics, and GSIS in pancreatic beta cells.
Main Methods:
- Western blotting to confirm VAV2 expression in beta cells and islets.
- siRNA-mediated knockdown of VAV2 to assess its impact on GSIS and Rac1 activation.
- Pharmacological inhibition of the VAV2-Rac1 interaction using Ehop-016.
- Confocal microscopy and live-cell imaging with LifeAct-GFP to analyze protein co-localization and actin remodeling.
Main Results:
- VAV2 is expressed in pancreatic beta cells (INS-1 832/13) and primary islets.
- VAV2 knockdown or inhibition of VAV2-Rac1 interaction significantly reduced glucose-induced Rac1 activation and GSIS.
- High glucose promoted Rac1 and VAV2 co-localization, and Ehop-016 inhibited glucose-induced actin remodeling.
Conclusions:
- VAV2 plays a critical role in mediating glucose-induced Rac1 activation and actin remodeling, which are essential for GSIS in pancreatic beta cells.
- VAV2 is a novel regulator of insulin secretion, highlighting its potential as a therapeutic target for diabetes.
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