Glucose-induced Rac1 Activation Research Articles

Multiple Guanine Nucleotide Exchange Factors Mediate Glucose-Induced Rac1 Activation and Insulin Secretion: Is It Precise Regulatory Control or a Case of Two Peas from the Same Pod?

Glucose-stimulated insulin secretion involves G protein (Rac1)-mediated cytoskeletal remodeling and vesicular transport and fusion with the plasma membrane. Recent evidence implicates at least three guanine nucleotide exchange factors (GEFs), namely, Tiam1, Vav2, and P-Rex1, in glucose-induced activation of Rac1 and insulin secretion. This Viewpoint highlights potential mechanisms underlying Tiam1/Vav2/P-Rex1 sensitive Rac1-mediated insulin secretion in the glucose-stimulated β-cell.

P-Rex1 Mediates Glucose-Stimulated Rac1 Activation and Insulin Secretion in Pancreatic β-Cells.

Despite the published evidence implicating phosphoinositide 3-kinase (PI3-kinase) in the regulation of islet function, limited information is available on the putative contributory roles of its downstream signaling steps, including the phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchange factor 1 (P-Rex1) signaling pathway in the islet β-cell. Therefore, we investigated potential roles for P-Rex1 in glucose-stimulated Rac1 activation and insulin secretion in insulin-secreting (INS-1 832/13) β-cells. Glucose-stimulated Insulin secretion (GSIS) was quantified by ELISA. Expression of endogenous P-Rex1 and RhoG was suppressed by siRNA transfection using the DharmaFect1 reagent. Total membrane and cytosolic fractions were isolated using the Mem-PER Plus Membrane Extraction Kit. The degree of activation of Rac1 was determined by the pull-down assay. P-Rex1 is expressed in INS-1 832/13 cells, normal rat islets and human islets. siRNA-mediated knockdown of P-Rex1 attenuated glucose-induced Rac1 activation, membrane association and insulin secretion. RhoG, which has been implicated in PI3-kinase-mediated Rac1 activation in other cell types, appears not to contribute to GSIS since the siRNA-mediated knockdown of RhoG failed to exert significant effects on GSIS. LY294002, a known inhibitor of PI3-kinase, potentiated GSIS without affecting glucose-induced Rac1 activation. Based on these findings, we conclude that P-Rex1 plays a novel regulatory role in glucose-induced Rac1 activation and insulin secretion.

Adaptor Protein p66Shc: A Link Between Cytosolic and Mitochondrial Dysfunction in the Development of Diabetic Retinopathy.

Diabetes increases oxidative stress in the retina and dysfunctions their mitochondria, accelerating capillary cell apoptosis. A 66 kDa adaptor protein, p66Shc, is considered as a sensor of oxidative stress-induced apoptosis. In the pathogenesis of diabetic retinopathy, a progressive disease, reactive oxygen species (ROS) production by activation of a small molecular weight G-protein (Ras-related C3 botulinum toxin substrate 1 [Rac1])-Nox2 signaling precedes mitochondrial damage. Rac1 activation is facilitated by guanine exchange factors (GEFs), and p66Shc increases Rac1-specific GEF activity of Son of Sevenless 1 (Sos1). p66Shc also possesses oxidoreductase activity and can directly stimulate mitochondrial ROS generation. Our aim was to investigate the role of p66Shc in the development of diabetic retinopathy and mechanism of its transcription. High glucose increased p66Shc expression in human retinal endothelial cells, and elevated acetylated histone 3 lysine 9 (H3K9) levels and transcriptional factor p53 binding at its promoter. Glucose also augmented interactions between Rac1 and Sos1 and activated Rac1-Nox2. Phosphorylation of p66Shc was increased, allowing it to interact with peptidyl prolyl isomerase to facilitate its localization inside the mitochondria, culminating in mitochondrial damage. P66shc-small interfering RNA (siRNA) inhibited glucose-induced Rac1 activation and mitochondrial damage. Similar results are observed in retinal microvessels from diabetic rats. This is the first report identifying the role of p66Shc in the development of diabetic retinopathy and implicating increased histone acetylation in its transcriptional regulation. Thus, p66Shc has dual role in the development of diabetic retinopathy; its regulation in the early stages of the disease should impede Rac1-ROS production and, in the later stages, prevent mitochondrial damage and initiation of a futile cycle of free radicals.

EHT 1864, a small molecule inhibitor of Ras-related C3 botulinum toxin substrate 1 (Rac1), attenuates glucose-stimulated insulin secretion in pancreatic β-cells

Glucose-stimulated insulin secretion (GSIS) in the pancreatic β-cells entails a variety of signaling mechanisms including activation of small GTP-binding proteins (G-proteins). Previous studies from our laboratory in human islets, rodent islets and clonal β-cells have demonstrated that G-proteins (e.g., Arf6, Cdc42 and Rac1) play novel roles in cytoskeletal remodeling, which is a critical step in the trafficking of insulin-laden secretory granules for fusion with plasma membrane and release of insulin. To further understand regulatory roles of Rac1 in GSIS, we utilized, herein, EHT 1864, a small molecule inhibitor, which attenuates Rac1 activation by retaining the G-protein in an inert/inactive state, thereby preventing activation of its downstream effector proteins. We demonstrate that EHT 1864 markedly attenuated GSIS in INS-1 832/13 cells. In addition, EHT 1864 significantly reduced glucose-induced activation and membrane targeting of Rac1 in INS-1 832/13 cells. This Rac1 inhibitor also suppressed glucose-induced activation of ERK1/2 and p53, but not Akt. Lastly, unlike the inhibitors of protein prenylation (simvastatin), EHT 1864 did not exert any significant effects on cell morphology (cell rounding) under the conditions it attenuated Rac1-sensitive signaling steps leading to GSIS. Based on these findings, we conclude that EHT 1864 specifically inhibits glucose-induced Rac1 activation and membrane association and associated downstream signaling events culminating in inhibition of GSIS.

Isoprenylcysteine carboxyl methyltransferase facilitates glucose-induced Rac1 activation, ROS generation and insulin secretion in INS 832/13 β-cells

Isoprenylcysteine carboxyl methyltransferase (ICMT) catalyzes the post-translational methylation of C-terminal cysteines of isoprenylated proteins, including small G-proteins and the γ-subunits of heterotrimeric G-proteins. It is widely felt that carboxymethylation promotes efficient membrane association of the methylated proteins and specific protein-protein interactions. In the current study, we tested the hypothesis that ICMT-mediated carboxymethylation of specific proteins (e.g., Rac1) plays a regulatory role in glucose-stimulated insulin secretion (GSIS). Western blot analysis indicated that lCMT is expressed and predominantly membrane associated in INS 832/13 β-cells. siRNA-mediated knockdown of endogenous expression of ICMT markedly attenuated glucose, but not KCl-induced insulin secretion. These findings were further supported by pharmacological observations, which suggested a marked reduction in glucose-, but not KCl-stimulated insulin secretion by acetyl farnesyl cysteine (AFC), a selective inhibitor of ICMT. In addition, glucose-induced Rac1 activation, a hallmark signaling step involved in glucose-stimulated insulin secretion, was markedly inhibited following pharmacological (AFC) or molecular biological (siRNA-ICMT) inhibition of ICMT. Lastly, we also noticed a marked reduction in glucose-induced acute increase in the generation of reactive oxygen species in INS 832/13 cells pre-treated with AFC or transfected with siRNA-ICMT. Together, these data suggest that ICMT regulates glucose-induced Rac1 activation, generation of reactive oxygen species and insulin secretion in pancreatic β-cells.

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

OBJECTIVEPosttranslational 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 Raf-1/extracellular signal–related kinase (ERK1/2) signaling pathway in glucose-induced Rac1 activation and insulin secretion in the pancreatic β-cell.RESEARCH DESIGN AND METHODSThese 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 glucose-mediated activation of ERK1/2, Rac1, and insulin secretion. Activation of ERK1/2 was determined by Western blotting. Rac1 activation (i.e., Rac1.GTP) was quantitated by p21-activated kinase pull-down assay. Insulin release was quantitated by enzyme-linked immunosorbent assay.RESULTSCoprovision of structure-specific inhibitors of farnesyl transferase (FTase; e.g., FTI-277 or FTI-2628) or siRNA-mediated knockdown of FTase β-subunit resulted in a significant inhibition of glucose-stimulated ERK1/2 and Rac1 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 Rac1 activation leading to glucose-induced insulin release. Lastly, siRNA-mediated silencing of endogenous expression of ERK1/2 markedly attenuated glucose-induced Rac1 activation and insulin secretion.CONCLUSIONSTogether, 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.

Glucose-stimulated Cdc42 Signaling Is Essential for the Second Phase of Insulin Secretion

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 second phase 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 beta-cells. Cdc42 activation occurred early in secretion (3 min), whereas Rac1 activation required approximately 15-20 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 glucose-stimulated 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 beta-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.