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

SUBMITTER: Veluthakal R

PROVIDER: S-EPMC4591202 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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

Veluthakal Rajakrishnan R Tunduguru Ragadeepthi R Arora Daleep Kumar DK Sidarala Vaibhav V Syeda Khadija K Vlaar Cornelis P CP Thurmond Debbie C DC Kowluru Anjaneyulu A

Diabetologia 20150731 11

<h4>Aims/hypothesis</h4>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 (V ...[more]

PMID: 26224100

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Project description:Lorcaserin is a serotonergic agonist specific to the 5-hydroxytryptamine 2c receptor (5-HT2CR) that is FDA approved for the long-term management of obesity with or without at least one weight-related comorbidity. Lorcaserin can restrain patients' appetite and improve insulin sensitivity and hyperinsulinemia mainly through activating 5-HT2CR in the hypothalamus. It is known that the mCPP, a kind of 5-HT2CR agonist, decreases plasma insulin concentration in mice and previous research in our laboratory found that mCPP inhibited glucose-stimulated insulin secretion (GSIS) by activating 5-HT2CR on the β cells. However, the effect of lorcaserin on GSIS of pancreatic β cell has not been studied so far. The present study found that 5-HT2CR was expressed in both mouse pancreatic β cells and β-cell-derived MIN6 cells. Dose-dependent activation of 5-HT2CR by lorcaserin suppressed GSIS and SB242084 or knockdown of 5-HT2CR abolished lorcaserin's effect in vitro. Additionally, lorcaserin also suppressed GSIS in high-fat diet (HFD)-fed mice in dose-dependent manner. Lorcaserin did not change insulin synthesis ATP content, but lorcaserin decrease cytosolic free calcium level [(Ca2+)i] in MIN6 cells stimulated with glucose and also inhibit insulin secretion and (Ca2+)i in MIN6 treated with potassium chloride. Furthermore, stimulation with the L-type channel agonist, Bay K8644 did not restore GSIS in MIN6 exposed to lorcaserin. Lorcaserin inhibits the cAMP generation of MIN6 cells and pretreatment with the Gα i/o inhibitor pertussis toxin (PTX), abolished lorcaserin-induced suppression of GSIS in β cells, while membrane-permeable cAMP analogue db-cAMP had same effect as PTX. These date indicated lorcaserin coupled to PTX-sensitive Gα i/o proteins in β cells reduced intracellular cAMP level and Ca2+ influx, thereby causing GSIS dysfunction of β cell. These results highlight a novel signaling mechanism of lorcaserin and provide valuable insights into the further investigation of 5-HT2CR functions in β-cell biology and it also provides guidance for the clinical application of lorcaserin.

Dataset Information

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

Publications

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

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