VAV2, a guanine nucleotide exchange factor for Rac1, regulates glucose-stimulated insulin secretion in pancreatic beta cells

Abstract

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 Our data provide the first evidence to implicate VAV2 in glucose-induced Rac1 activation, actin remodelling and GSIS in pancreatic beta cells.

Inhibition of Prenylation Promotes Caspase 3 Activation, Lamin B Degradation and Loss in Metabolic Cell Viability in Pancreatic β-Cells

“…Furthermore, incubation of INS-1 832/13 cells with Simvastatin (15 or 30 µM), but not the diluent (DMSO) induced clear morphological changes (cell rounding) in these cells (Fig. 2; Panel C); these data further validate the original hypothesis that protein prenylation plays significant regulatory roles in cell morphology and cytoskeletal arrangements and architecture [24, 25]. Data depicted in Fig.…”

“…We next assessed alterations in the distribution of active caspase 3 and degraded lamin B in INS-1 832/13 cells exposed to either basal glucose or FTI-277. Total hydrophobic and hydrophilic compartments from these cells were isolated using the Triton X-114 phase separation method as we described in [23, 24]. Data in Fig.…”

“…*P<0.05 vs. LG. Panel D: Lysates from INS-1 832/13 cells treated with low glucose (2.5 mM) in the absence and presence of FTI-277 (10 µM) were subjected to a single-step centrifugation to separate the supernatant cytosolic (Cyto) and the membrane-pellet (Mem) fractions followed by hydrophobic (HPB) and hydrophilic (HPL) phase partitioning with Triton X-114 detergent [23, 24]. The lysate proteins were resolved by SDS-PAGE and transferred to a nitrocellulose membrane.…”

“…Cell lysates were homogenized in a homogenization buffer (20 mM Tris-HCl, pH 7.5, 0.5 mM EGTA, 2 mM MgCl 2 and protease inhibitor cocktail) and subjected to a single-step centrifugation at 100, 000 g for 60 min at 4°C in order to separate the cytosol (supernatant) and the membrane (pellet) fractions. The membrane fraction was then subjected to hydrophobic and hydrophilic phase partitioning using Triton X-114 according to the method we described in [23, 24]. …”

Tiam1/Vav2-Rac1 axis: A tug-of-war between islet function and dysfunction

“…The GDP-bound inactive Rac1 is converted to its active, GTP-bound conformation by GEFs, including T-lymphocyte invasive and metastasis protein 1 [Tiam1] and the guanine nucleotide exchange factor Vav2 [Vav2]. Published evidence suggests that both Tiam1 and Vav2 are expressed in a variety of insulin-secreting cells including clonal INS-1 832/13 cells, rodent islets and human islets [9,10]. Using small molecule inhibitors for Tiam1 [e.g., NSC23766] and Vav2 [e.g., Ehop-016] recent studies have demonstrated critical regulatory roles for these GEFs in islet function in health and metabolic stress [see below].…”