Zhanxiang Wang

Mechanisms of biphasic insulin-granule exocytosis – roles of the cytoskeleton, small GTPases and SNARE proteins

The release of insulin from pancreatic islets requires negative regulation to ensure low levels of insulin release under resting conditions, as well as positive regulation to facilitate robust responsiveness to conditions of elevated fuel or glucose. The first phase of release involves the plasma-membrane fusion of a small pool of granules, termed the readily releasable pool; these granules are already at the membrane under basal conditions, and discharge their cargo in response to nutrient and also non-nutrient secretagogues. By contrast, second-phase secretion is evoked exclusively by nutrients, and involves the mobilization of intracellular granules to t-SNARE sites at the plasma membrane to enable the distal docking and fusion steps of insulin exocytosis. Nearly 40 years ago, the actin cytoskeleton was first recognized as a key mediator of biphasic insulin release, and was originally presumed to act as a barrier to block granule docking at the cell periphery. More recently, however, the discovery of cycling GTPases that are involved in F-actin reorganization in the islet β-cell, combined with the availability of reagents that are more specific and tools with which to study the mechanisms that underlie granule movement, have contributed greatly to our understanding of the role of the cytoskeleton in regulating biphasic insulin secretion. Herein, we provide historical perspective and review recent progress that has been made towards integrating cytoskeletal reorganization and cycling of small Rho-, Rab- and Ras-family GTPases into our current models of stimulus-secretion coupling and second-phase insulin release.

Filamentous Actin Regulates Insulin Exocytosis through Direct Interaction with Syntaxin 4

Glucose-induced insulin exocytosis is coupled to associations between F-actin and SNARE proteins, although the nature and function of these interactions remains unknown. Toward this end we show here that both Syntaxin 1A and Syntaxin 4 associated with F-actin in MIN6 cells and that each interaction was rapidly and transiently diminished by stimulation of cells with d-glucose. Of the two isoforms, only Syntaxin 4 was capable of interacting directly with F-actin in an in vitro sedimentation assay, conferred by the N-terminal 39-112 residues of Syntaxin 4. The 39-112 fragment was capable of selective competitive inhibitory action, disrupting endogenous F-actin-Syntaxin 4 binding in MIN6 cells. Disruption of F-actin-Syntaxin 4 binding correlated with enhanced glucose-stimulated insulin secretion, mediated by increased granule accumulation at the plasma membrane and increased Syntaxin 4 accessibility under basal conditions. However, no increase in basal level Syntaxin 4-VAMP2 association occurred with either latrunculin treatment or expression of the 39-112 fragment. Taken together, these data disclose a new underlying mechanism by which F-actin negatively regulates exocytosis via binding and blocking Syntaxin 4 accessibility, but they also reveal the existence of additional signals and/or steps required to trigger the subsequent docking and fusion steps of exocytosis.

Inhibition or Ablation of p21-activated Kinase (PAK1) Disrupts Glucose Homeostatic Mechanisms in Vivo

Background: P21-activated kinase (PAK1) is a downstream effector of the GTPase Cdc42. Results: Inhibition of Cdc42-PAK1 signaling in human islets inhibited insulin secretion. PAK1 knock-out mice showed defects in insulin release and skeletal muscle insulin action, underlying impaired whole body glucose homeostasis. Conclusion: Attenuated PAK1 abundance/activation may contribute to type 2 diabetes susceptibility. Significance: Cdc42-PAK1 signaling is crucial for regulating glucose homeostasis in vivo. The p21-activated kinase PAK1 is implicated in tumorigenesis, and efforts to inhibit PAK1 signaling as a means to induce tumor cell apoptosis are underway. However, PAK1 has also been implicated as a positive effector of mechanisms in clonal pancreatic beta cells and skeletal myotubes that would be crucial to maintaining glucose homeostasis in vivo. Of relevance, human islets of Type 2 diabetic donors contained ∼80% less PAK1 protein compared with non-diabetics, implicating PAK1 in islet signaling/scaffolding functions. Mimicking this, islets from PAK1−/− knock-out mice exhibited profound defects in the second/sustained-phase of insulin secretion. Reiteration of this specific defect by human islets treated with the PAK1 signaling inhibitor IPA3 revealed PAK1 signaling to be of primary functional importance. Analyses of human and mouse islet beta cell signaling revealed PAK1 activation to be 1) dependent upon Cdc42 abundance, 2) crucial for signaling downstream to activate ERK1/2, but 3) dispensable for cofilin phosphorylation. Importantly, the PAK1−/− knock-out mice were found to exhibit whole body glucose intolerance in vivo. Exacerbating this, the PAK1−/− knock-out mice also exhibited peripheral insulin resistance. Insulin resistance was coupled to ablation of insulin-stimulated GLUT4 translocation in skeletal muscle from PAK1−/− knock-out mice, and in sharp contrast to islet beta cells, skeletal muscle PAK1 loss was underscored by defective cofilin phosphorylation but normal ERK1/2 activation. Taken together, these data provide the first human islet and mammalian in vivo data unveiling the key and crucial roles for differential PAK1 signaling in the multi-tissue regulation of whole body glucose homeostasis.

Differential phosphorylation of RhoGDI mediates the distinct cycling of Cdc42 and Rac1 to regulate second-phase insulin secretion.

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 interference-depleted 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.

A p21-activated kinase (PAK1) signaling cascade coordinately regulates F-actin remodeling and insulin granule exocytosis in pancreatic β cells.

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.

Gelsolin Associates with the N Terminus of Syntaxin 4 to Regulate Insulin Granule Exocytosis

The plasma membrane soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) protein syntaxin (Syn)4 is required for biphasic insulin secretion, although how it regulates each phase remains unclear. In a screen to identify new Syn4-interacting factors, the calcium-activated F-actin-severing protein gelsolin was revealed. Gelsolin has been previously implicated as a positive effector of insulin secretion, although a molecular mechanism to underlie this function is lacking. Toward this, our in vitro binding studies showed the Syn4-gelsolin interaction to be direct and mediated by the N-terminal Ha domain (amino acid residues 39-70) of Syn4. Syn4-gelsolin complexes formed under basal conditions and dissociated upon acute glucose or KCl stimulation; nifedipine blocked dissociation. The dissociating action of secretagogues could be mimicked by expression of the N-terminal Ha domain of Syn4 fused to green fluorescent protein (GFP) (GFP-39-70). Furthermore, GFP-39-70 expression in isolated mouse islet and clonal MIN6 β-cells initiated insulin release in the absence of appropriate stimuli. Consistent with this, the inhibitory GFP-39-70 peptide also initiated Syn4 activation in the absence of stimuli. Moreover, although MIN6 β-cells expressing the GFP-39-70 peptide maintained normal calcium influx in response to KCl, KCl-stimulated insulin secretion and the triggering pathway of insulin secretion were significantly impaired. Taken together, these data support a mechanistic model for gelsolins role in insulin exocytosis: gelsolin clamps unsolicited soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE)-regulated exocytosis through direct association with Syn4 in the absence of appropriate stimuli, which is relieved upon stimulus-induced calcium influx to activate gelsolin and induce its dissociation from Syn4 to facilitate insulin exocytosis.

Cool-1/βPIX functions as a guanine nucleotide exchange factor in the cycling of Cdc42 to regulate insulin secretion

Second-phase insulin release requires the sustained mobilization of insulin granules from internal storage pools to the cell surface for fusion with the plasma membrane. However, the detailed mechanisms underlying this process remain largely unknown. GTP-loading of the small GTPase Cdc42 is the first glucose-specific activation step in the process, although how glucose triggers Cdc42 activation is entirely unknown. In a directed candidate screen for guanine nucleotide exchange factors (GEFs), which directly activate small GTPases, Cool-1/βPix was identified in pancreatic islet beta cells. In support of its role as the beta cell Cdc42 GEF, βPix coimmunoprecipitated with Cdc42 in human islets and MIN6 beta cells in a glucose-dependent manner, peaking just prior to Cdc42 activation. Furthermore, RNAi-mediated βPix reduction by 50% corresponded to full ablation of glucose-induced Cdc42 activation and significant attenuation of basal and glucose-stimulated insulin secretion. Of the two Cdc42 guanine nucleotide dissociation inhibitor (GDI) proteins identified in beta cells, βPix competed selectively with caveolin-1 (Cav-1) but not RhoGDI in coimmunoprecipitation and GST-Cdc42-GDP interaction assays. However, a phospho-deficient Cav-1-Y14F mutant failed to compete with βPix; Cav-1(Tyr14) is an established phosphorylation site for Src kinase. Taken together, these data support a new model, wherein glucose stimulates Cav-1 and induces its dissociation from Cdc42, possibly via Src kinase activation to phosphorylate Cav-1(Tyr14), to promote Cdc42-βPix binding and Cdc42 activation, and to trigger downstream signaling and ultimately sustain insulin release.

YES, a Src family kinase, is a proximal glucose-specific activator of cell division cycle control protein 42 (Cdc42) in pancreatic islet β cells.

Background: Although Cdc42 signaling is a known requirement for insulin secretion to occur, how it is initiated remains unknown. Results: YES kinase is required for Cdc42 activation in human islets and β cells. Conclusion: YES is a proximal, glucose-specific activator of Cdc42 and glucose-simulated insulin secretion. Significance: YES may provide a novel target for improvement of functional β cell mass. Second-phase insulin secretion sustains insulin release in the face of hyperglycemia associated with insulin resistance, requiring the continued mobilization of insulin secretory granules to the plasma membrane. Cdc42, the small Rho family GTPase recognized as the proximal glucose-specific trigger to elicit second-phase insulin secretion, signals downstream to activate the p21-activated kinase (PAK1), which then signals to Raf-1/MEK/ERK to induce filamentous actin (F-actin) remodeling, to ultimately mobilize insulin granules to the plasma membrane. However, the steps required to initiate Cdc42 activation in a glucose-specific manner in β cells have remained elusive. Toward this, we identified the involvement of the Src family kinases (SFKs), based upon the ability of SFK inhibitors to block glucose-stimulated Cdc42 and PAK1 activation events as well as the amplifying pathway of glucose-stimulated insulin release, in MIN6 β cells. Indeed, subsequent studies performed in human islets revealed that SFK phosphorylation was induced only by glucose and within 1 min of stimulation before the activation of Cdc42 at 3 min. Furthermore, pervanadate treatment validated the phosphorylation event to be tyrosine-specific. Although RT-PCR showed β cells to express five different SFK proteins, only two of these, YES and Fyn kinases, were found localized to the plasma membrane, and of these two, only YES kinase underwent glucose-stimulated tyrosine phosphorylation. Immunodetection and RNAi analyses further established YES kinase as a proximal glucose-specific signal in the Cdc42-signaling cascade. Identification of YES kinase provides new insight into the mechanisms underlying the sustainment of insulin secretion via granule mobilization/replenishment and F-actin remodeling.

PAK1 limits the expression of the pro‐apoptotic protein Bad in pancreatic islet β‐cells

Human type 2 diabetes is associated with β‐cell apoptosis, and human islets from diabetic donors are ∼80% deficient in PAK1 protein. Toward addressing linkage of PAK1 to β‐cell survival, PAK1–siRNA targeted MIN6 pancreatic β‐cells were found to exhibit increased caspase‐3 cleavage, cytosolic cytochrome‐C and the pro‐apoptotic protein Bad. PAK1+/− heterozygous mouse islets recapitulated the upregulation of Bad protein expression, as did hyperglycemic treatment of human or mouse islets; Bad levels were exacerbated most in PAK1+/− islets subjected to hyperglycemic stress. These data implicate PAK1 in β‐cell survival via quenching of Bad protein expression, and suggest PAK1 as potential molecular target to preserve β‐cell mass.