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The small Rho family GTPases Cdc42 and Rac1 have each been shown to function in insulin exocytosis and are presumed to function in actin remodeling and insulin granule mobilization. However, whether either GTPase is required for the mobilization phase of insulin release (second phase) and are linked in a common signaling pathway has remained unknown. Here we demonstrate that small interfering RNA-mediated depletion of Cdc42 from isolated islets results in the selective loss of secondphase insulin release. Consistent with a role in this nutrient-dependent phase, Cdc42 activation was detected exclusively in response to D-glucose and was unresponsive to KCl or non-metabolizable glucose analogs in MIN6 ␤-cells. Cdc42 activation occurred early in secretion (3 min), whereas Rac1 activation required ϳ15-20 min, suggesting Cdc42 as proximal and Rac1 as distal regulators of second-phase secretion. Importantly, Rac1 activation and function was linked in a common pathway downstream of Cdc42; Cdc42 depletion ablated glucose-induced Rac1 activation, and expression of constitutively active Rac1 in Cdc42-depleted cells functionally restored glucosestimulated insulin secretion. Occurring at a time midway between Cdc42 and Rac1 activations was the phosphorylation of p21-activated-kinase 1 (Pak1), and this phosphorylation event required Cdc42. Moreover, small interfering RNA-mediated Pak1 depletion abolished Rac1 activation and glucose-stimulated insulin release, suggesting that Pak1 may mediate the link between Cdc42 and Rac1 in this pathway. Taken together, these data substantiate the existence of a novel signaling pathway in the islet ␤-cell whereby Cdc42 functions as a key proximal transmitter of the glucose signal early in stimulus-secretion coupling to support the later stage of insulin release.Insulin is released from pancreatic islet ␤-cells in two distinct phases in response to glucose stimulation. The first phase of insulin release can be elicited by elevation of intracellular calcium levels to trigger fusion of granules pre-docked at the plasma membrane, a glucose stimulation. The second phase of insulin release requires the amplifying action of glucose and is presumed to require mobilization of storage pool granules to the cell surface to sustain insulin release for an hour or longer depending upon how long the ␤-cell detects stimulatory levels of glucose. Thousands of storage granules exist behind a filamentous actin (F-actin) barrier in the pancreatic ␤-cell, and F-actin remodeling is known to mobilize granules to the target membrane soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) 2 sites at the cell surface, yet the mechanisms involved in remodeling and granule mobilization are largely unknown and untested.We and others have presented evidence to suggest that the key to actin remodeling in the islet ␤-cell may lie in the glucosespecific activation of the small Rho family GTPase protein Cdc42 (1-5). We have recently demonstrated that Cdc42 is kept in its GDP-loaded and inactivated sta...

Glucose-induced insulin exocytosis is coupled to associationsInsulin granules are exocytosed in two distinct phases. Firstphase insulin granule release involves the rapid fusion of a small pool of granules that are already present at the plasma membrane under basal conditions, termed the readily releasable pool, and these granules will discharge their cargo in response to nutrient and also non-nutrient secretagogues (1-4). In contrast, second-phase secretion is evoked only in response to nutrients and involves additional steps such as the mobilization of intracellular storage granules, targeting of granules to SNARE 3 sites for docking and fusion steps of exocytosis and insulin release. Remarkably, although the actin cytoskeleton has been considered to play a principle regulatory role in glucose-stimulated insulin secretion since 1968 (5, 6), the lack of mechanistic data has impeded the inclusion of cytoskeletal input into models detailing the regulation of biphasic insulin release. F-actin was originally shown to function as a "cell web" in islet beta cells (7-10). This notion was consistent with observations from other cell types and led to the concept that cytoskeletal disruption serves as a mechanism to clear away the F-actin barrier and permit access of granules to the plasma membrane (11)(12)(13)(14). However this model did not suffice to explain why insulin granule transport still requires F-actin as a motive force (9, 15), nor did it explain why F-actin is both increased and decreased by glucose (16 -19). Distinct from neurotransmitter exocytosis or GLUT4 translocation events that are impacted by F-actin, insulin release occurs over a long time period and in discrete phases, requiring the readily releasable pool of granules at the plasma membrane to be refilled from the more intracellular storage pool in a carefully metered manner. More recently, glucose-induced F-actin remodeling in beta cells has been visualized by dynamic changes in cortical F-actin (20 -23). No changes in the F/G-actin ratio were detected, indicating that the F-actin was being remodeled and reorganized as opposed to a general loss of cellular F-actin content in response to glucose. Remodeling occurred specifically in response to D-glucose, and not L-glucose or KCl stimulation, and was coupled to SNARE protein-mediated exocytosis.Insulin granule exocytosis requires two syntaxin isoforms: Syntaxin 1A and Syntaxin 4. Syntaxin 1A null mouse islets show significantly fewer predocked granules, which support firstphase insulin release in response to calcium influx, although second-phase secretion is normal (24). Syntaxin 4 heterozygous knock-out mouse islets show decreased first-phase secretion and also a slight decrease in second-phase secretion (25). In addition, Syntaxin 4 overexpressing transgenic mouse islets have enhanced second-phase secretion (25). Other cell systems that utilize multiple syntaxins show partitioning of interactions and localization to particular membrane compartments to achieve differential modes of vesicle targetin...

Human islet studies implicate an important signaling role for the Cdc42 effector protein p21-activated kinase (PAK1) in the sustained/second-phase of insulin secretion. Because human islets from type 2 diabetic donors lack ~80% of normal PAK1 protein levels, the mechanistic requirement for PAK1 signaling in islet function was interrogated. Similar to MIN6 β cells, human islets elicited glucose-stimulated PAK1 activation that was sensitive to the PAK1 inhibitor, IPA3. Given that sustained insulin secretion has been correlated with glucose-induced filamentous actin (F-actin) remodeling, we tested the hypothesis that a Cdc42-activated PAK1 signaling cascade is required to elicit F-actin remodeling to mobilize granules to the cell surface. Live-cell imaging captured the glucose-induced cortical F-actin remodeling in MIN6 β cells; IPA3-mediated inhibition of PAK1 abolished this remodeling. IPA3 also ablated glucose-stimulated insulin granule accumulation at the plasma membrane, consistent with its role in sustained/second-phase insulin release. Both IPA3 and a selective inhibitor of the Cdc42 GTPase, ML-141, blunted the glucose-stimulated activation of Raf-1, suggesting Raf-1 to be downstream of Cdc42→PAK1. IPA3 also inhibited MEK1/2 activation, implicating the MEK1/2→ERK1/2 cascade to occur downstream of PAK1. Importantly, PD0325901, a new selective inhibitor of MEK1/2→ERK1/2 activation, impaired F-actin remodeling and the sustained/amplification pathway of insulin release. Taken together, these data suggest that glucose-mediated activation of Cdc42 leads to activation of PAK1 and prompts activation of its downstream targets Raf-1, MEK1/2 and ERK1/2 to elicit F-actin remodeling and recruitment of insulin granules to the plasma membrane to support the sustained phase of insulin release.

Cdc42 cycling through GTP/GDP states is critical for its function in the second/granule mobilization phase of insulin granule exocytosis in pancreatic islet beta cells, although the identities of the Cdc42 cycling proteins involved remain incomplete. Using a tandem affinity purification-based mass spectrometry screen for Cdc42 cycling factors in beta cells, RhoGDI was identified. RNA interference-mediated depletion of RhoGDI from isolated islets selectively amplified the second phase of insulin release, consistent with the role of RhoGDI as a Cdc42 cycling factor. Replenishment of RhoGDI to RNA interferencedepleted cells normalized secretion, confirming the action of RhoGDI to be that of a negative regulator of Cdc42 activation. Given that RhoGDI also regulates Rac1 activation in beta cells, and that Rac1 activation occurs in a Cdc42-dependent manner, the question as to how the beta cell utilized RhoGDI for differential Cdc42 and Rac1 cycling was explored. Co-immunoprecipitation was used to determine that RhoGDI-Cdc42 complexes dissociated upon stimulation of beta cells with glucose for 3 min, correlating with the timing of glucose-induced Cdc42 activation and the onset of RhoGDI tyrosine phosphorylation. Glucose-induced disruption of RhoGDI-Rac1 complexes occurred subsequent to this, coincident with Rac1 activation, which followed the onset of RhoGDI serine phosphorylation. RhoGDI-Cdc42 complex dissociation was blocked by mutation of RhoGDI residue Tyr-156, whereas RhoGDI-Rac1 dissociation was blocked by RhoGDI mutations Y156F and S101A/S174A. Finally, expression of a triple Y156F/S101A/S174A-RhoGDI mutant specifically inhibited only the second/granule mobilization phase of glucose-stimulated insulin secretion, overall supporting the integration of RhoGDI into the activation cycling mechanism of glucose-responsive small GTPases.