Protein Farnesylation-Dependent Raf/Extracellular Signal-Related Kinase Signaling Links to Cytoskeletal Remodeling to Facilitate Glucose-Induced Insulin Secretion in Pancreatic β-Cells

OBJECTIVE-Posttranslational prenylation (e.g., farnesylation) of small G-proteins is felt to be requisite for cytoskeletal remodeling and fusion of secretory vesicles with the plasma membrane. Here, we investigated roles of protein farnesylation in the signaling steps involved in Ral-1/extracellular signal-related kinase (ERK1/2) signaling pathway in glucose-induced Rac1 activation and insulin secretion in the pancreatic beta-cell. RESEARCH DESIGN AND METHODS-These studies were carried out in INS 832/13 cells and normal rat islets. Molecular biological (e.g., overexpression or small interfering RNA [siRNA]-mediated knockdown) and pharmacologic approaches were used to determine roles for farnesylation in glucosemediated activation of ERK1/2, Rac1, and insulin secretion. Activation of ERK1/2 was determined by Western blotting. Racl activation (i.e., Rac1.GTP) was quantitated by p21-activated kinase pull-down assay. Insulin release was quantitated by enzyme-linked immunosorbent assay. RESULTS-Coprovision of structure-specific inhibitors of farnesyl transferase (FTase; e.g., FTI-277 or FTI-2628) or siRNA-mediated knockdown of FTase beta-subunit resulted in a significant inhibition of glucose-stimulated ERK1/2 and Racl activation and insulin secretion. Pharmacologic inhibition of Raf-1 kinase using GW-5074 markedly reduced the stimulatory effects of glucose on ERK1/2 phosphorylation, Rac1 activation, and insulin secretion, suggesting that Raf-1 kinase activation may be upstream to ERK1/2 and Racl activation leading to glucose-induced insulin release. Lastly, siRNA-mediated silencing of endogenous expression of ERK1/2 markedly attenuated glucose-induced Racl activation and insulin secretion. CONCLUSIONS-Together, our findings provide the first evidence of a role for protein farnesylation in glucose-mediated regulation of the Raf/ERK signaling pathway culminating in the activation of Rac1, which has been shown to be necessary for cytoskeletal reorganization and exocytotic secretion of insulin. Diabetes 59:967-977, 2010