Hyperglycemic Stress Induces Expression, Degradation, and Nuclear Association of Rho GDP Dissociation Inhibitor 2 (RhoGDIß) in Pancreatic ß-Cells.

Small G proteins (e.g., Rac1) play critical regulatory roles in islet ß-cell function in health (physiological insulin secretion) and in metabolic stress (cell dysfunction and demise). Multiple regulatory factors for these G proteins, such as GDP dissociation inhibitors (GDIs), have been implicated in the functional regulation of these G proteins. The current set of investigations is aimed at understanding impact of chronic hyperglycemic stress on the expression and subcellular distribution of three known isoforms of RhoGDIs (RhoGDIα, RhoGDIß, and RhoGDIγ) in insulin-secreting ß-cells. The data accrued in these studies revealed that the expression of RhoGDIß, but not RhoGDIα or RhoGDIγ, is increased in INS-1 832/13 cells, rat islets, and human islets. Hyperglycemic stress also promoted the cleavage of RhoGDIß, leading to its translocation to the nuclear compartment. We also report that RhoGDIα, but not RhoGDIγ, is associated with the nuclear compartment. However, unlike RhoGDIß, hyperglycemic conditions exerted no effects on RhoGDIα's association with nuclear fraction. Based on these observations, and our earlier findings of the translocation of Rac1 to the nuclear compartment under the duress of metabolic stress, we conclude that the RhoGDIß-Rac1 signaling module promotes signals from the cytosolic to the nucleus, culminating in accelerated ß-cell dysfunction under metabolic stress.

Novel regulatory roles of small G protein GDP dissociation stimulator (smgGDS) in insulin secretion from pancreatic ß-cells.

Emerging evidence implicates novel roles for small G protein GDP dissociation stimulator (smgGDS) in G protein activation and subsequent targeting to relevant subcellular compartments for effector regulation. Given the well-established roles of small G proteins in insulin secretion, we undertook this investigation to determine the putative roles of smgGDS in insulin secretion. Immunoblotting studies revealed that both splice variants of smgGDS are expressed in human islets, rat islets and INS-1 832/13 cells. A significant inhibition (-52%) of glucose-stimulated insulin secretion (GSIS) was observed in INS-1 832/13 cells following siRNA-mediated depletion of smgGDS. In addition, insulin secretion elicited by a membrane depolarizing concentration of KCl (via increased calcium influx), forskolin (via increased cAMP generation) or IBMX (via inhibition of phosphodiesterase) was inhibited by -49%, -27%, and -28%, respectively. Subcellular distribution studies revealed no significant alterations in the abundance of smgGDS in the cytosolic and membrane fractions during the 45-min exposure of INS-1 832/13 cells to an insulinotropic concentration of glucose. Together, we present the first evidence of expression of smgGDS in human islets, rodent islets, and clonal ß-cells. We also demonstrate novel regulatory roles of these proteins in insulin secretion derived from glucose metabolic events, including calcium- and cAMP-dependent signaling steps.

Regulatory Roles of Histone Deacetylation in Metabolic Stress-Induced Expression of Caspase Recruitment Domain-Containing Protein 9 (CARD9) in Pancreatic ß-Cells.

CARD9, a scaffolding protein, has been implicated in the pathogenesis of metabolic diseases, including obesity and diabetes. We recently reported novel roles for CARD9 in islet ß-cell dysregulation under duress of gluco (HG)- and glucolipotoxic (GLT) stress. CARD9 expression was also increased in ß-cells following exposure to HG and GLT stress. The current study is aimed at understanding the putative roles of histone deacetylation in HG- and GLT-induced expression of CARD9. Using two structurally distinct inhibitors of histone deacetylases (HDACs), namely trichostatin (TSA) and suberoylanilide hydroxamic acid (SAHA), we provide the first evidence to suggest that the increased expression of CARD9 seen under duress of HG and GLT stress is under the regulatory control of histone deacetylation. Interestingly, the expression of protein kinase Cδ (PKCδ), a known upstream regulator of CARD9 activation, is also increased under conditions of metabolic stress. However, it is resistant to TSA and SAHA, suggesting that it is not regulated via histone deacetylation. Based on these data, we propose that targeting the appropriate HDACs, which mediate the expression (and function) of CARD9, might be the next step to further enhance our current understanding of the roles of CARD9 in islet dysfunction under metabolic stress and diabetes.

Hyperglycemic Conditions Promote Rac1-Mediated Serine536 Phosphorylation of p65 Subunit of NFκB (RelA) in Pancreatic Beta Cells.

BACKGROUND/AIMS: We recently reported increased phosphorylation (at S536) of the p65 subunit of NFκB (Rel A) in pancreatic beta (INS-1 832/13) cells following exposure to hyperglycemic (HG) conditions. We also demonstrated that HG-induced S536 phosphorylation of p65 is downstream to the regulatory effects of CARD9 since deletion of CARD9 expression significantly attenuated HG-induced S536 phosphorylation of p65 in beta cells. The overall objective of the current investigation is to identify putative mechanisms underlying HG-induced phosphorylation of p65 in islet beta cells following exposure to HG conditions. METHODS: INS-1 832/13 cells were incubated in low glucose (LG; 2.5 mM) or high glucose (HG; 20 mM) containing media for 24 hours in the absence or presence of small molecule inhibitors of G protein prenylation and activation. Non-nuclear and nuclear fractions were isolated from INS-1 832/13 cells using a commercially available (NE-PER) kit. Degree of S536 phosphorylation of the p65 subunit was quantified by western blotting and densitometry. RESULTS: HG-induced p65 phosphorylation was significantly attenuated by inhibitors of protein prenylation (e.g., simvastatin and L-788,123). Pharmacological inhibition of Tiam1-Rac1 (e.g., NSC23766) and Vav2-Rac1 (e.g., Ehop-016) signaling pathways exerted minimal effects on HG-induced p65 phosphorylation. However, EHT-1864, a small molecule compound, which binds to Rac1 thereby preventing GDP/GTP exchange, markedly suppressed HG-induced p65 phosphorylation, suggesting that Rac1 activation is requisite for HG-mediated p65 phosphorylation. Lastly, EHT-1864 significantly inhibited nuclear association of STAT3, but not total p65, in INS-1 832/13 cells exposed to HG conditions. CONCLUSION: Activation of Rac1, a step downstream to HG-induced activation of CARD9, might represent a requisite signaling step in the cascade of events leading to HG-induced S536 phosphorylation of p65 and nuclear association of STAT3 in pancreatic beta cells. Data from these investigations further affirm the role(s) of Rac1 as a mediator of metabolic stress- induced dysfunction of the islet beta cell.

Underappreciated roles for Rho GDP dissociation inhibitors (RhoGDIs) in cell function: Lessons learned from the pancreatic islet ß-cell.

Rho subfamily of G proteins (e.g., Rac1) have been implicated in glucose-stimulated insulin secretion from the pancreatic ß-cell. Interestingly, metabolic stress (e.g., chronic exposure to high glucose) results in sustained activation of Rac1 leading to increased oxidative stress, impaired insulin secretion and ß-cell dysfunction. Activation-deactivation of Rho G proteins is mediated by three classes of regulatory proteins, namely the guanine nucleotide exchange factors (GEFs), which facilitate the conversion of inactive G proteins to their active conformations; the GTPase-activating proteins (GAPs), which convert the active G proteins to their inactive forms); and the GDP-dissociation inhibitors (GDIs), which prevent the dissociation of GDP from G proteins. Contrary to a large number of GEFs (82 members) and GAPs (69 members), only three members of RhoGDIs (RhoGDIα, RhoGDIß and RhoGDIγ) are expressed in mammalian cells.Even though relatively smaller in number, the GDIs appear to play essential roles in G protein function (e.g., subcellular targeting) for effector activation and cell regulation. Emerging evidence also suggests that the GDIs are functionally regulated via post-translational modification (e.g., phosphorylation) and by lipid second messengers, lipid kinases and lipid phosphatases. We highlight the underappreciated regulatory roles of RhoGDI-Rho G protein signalome in islet ß-cell function in health and metabolic stress. Potential knowledge gaps in the field, and directions for future research for the identification of novel therapeutic targets to loss of functional ß-cell mass under the duress of metabolic stress are highlighted.

Molecular characteristics of proteins within the mitochondrial Fe-S cluster assembly complex.

Iron-Sulfur (Fe-S) clusters are essential for life, as they are widely utilized in nearly every biochemical pathway. When bound to proteins, Fe-S clusters assist in catalysis, signal recognition, and energy transfer events, as well as additional cellular pathways including cellular respiration and DNA repair and replication. In Eukaryotes, Fe-S clusters are produced through coordinated activity by mitochondrial Iron-Sulfur Cluster (ISC) assembly pathway proteins through direct assembly, or through the production of the activated sulfur substrate used by the Cytosolic Iron-Sulfur Cluster Assembly (CIA) pathway. In the mitochondria, Fe-S cluster assembly is accomplished through the coordinated activity of the ISC pathway protein complex composed of a cysteine desulfurase, a scaffold protein, the accessory ISD11 protein, the acyl carrier protein, frataxin, and a ferredoxin; downstream events that accomplish Fe-S cluster transfer and delivery are driven by additional chaperone/delivery proteins that interact with the ISC assembly complex. Deficiency in human production or activity of Fe-S cluster containing proteins is often detrimental to cell and organism viability. Here we summarize what is known about the structure and functional activities of the proteins involved in the early steps of assembling [2Fe-2S] clusters before they are transferred to proteins devoted to their delivery. Our goal is to provide a comprehensive overview of how the ISC assembly apparatus proteins interact to make the Fe-S cluster which can be delivered to proteins downstream to the assembly event.

RhoG-Rac1 Signaling Pathway Mediates Metabolic Dysfunction of the Pancreatic Beta-Cells Under Chronic Hyperglycemic Conditions.

BACKGROUND/AIMS: Published evidence suggests regulatory roles for small G proteins (Cdc42 and Rac1) in glucose-stimulated insulin secretion (GSIS) from pancreatic beta-cells. More recent evidence suggests novel roles for these G proteins, specifically Rac1, in the induction of metabolic dysfunction of the islet beta-cell under the duress of a variety of stress conditions. However, potential upstream regulators of sustained activation of Rac1 have not been identified in the beta-cell. Recent studies in other cell types have identified RhoG, a small G protein, as an upstream regulator of Rac1 under specific experimental conditions. Herein, we examined putative roles for RhoG in islet beta-cell dysregulation induced by glucotoxic conditions. METHODS: Expression of RhoG or GDIγ was suppressed by siRNA transfection using the DharmaFect1 reagent. Subcellular fractions were isolated using NE-PER Nuclear and Cytoplasmic Extraction Reagent kit. The degree of activation of Rac1 was assessed using a pull-down assay kit. Extent of cell death was quantified using a Cell Death Detection ELISAplus kit. RESULTS: RhoG is expressed in human islets, rat islets, and clonal INS-1 832/13 cells. siRNA-RhoG markedly attenuated sustained activation of Rac1 and caspase-3 in INS-1 832/13 cells exposed to hyperglycemic conditions (20 mM; 24 hours). In a manner akin to Rac1, which has been shown to translocate to the nuclear fraction to induce beta-cell dysfunction under metabolic stress, a significant increase in the association of RhoG with the nuclear fraction was observed in beta-cells under the duress of metabolic stress. Interestingly, GDIγ, a known regulator of RhoG, remained associated with non-nuclear fraction under conditions RhoG and Rac1 translocated to the membrane. Lastly, siRNA-RhoG modestly attenuated pancreatic beta-cell demise induced by high glucose exposure conditions, but such an effect was not statistically significant. CONCLUSION: Based on these data we conclude that RhoG-Rac1 signaling module plays critical regulatory roles in promoting mitochondrial dysfunction (caspase-3 activation) of the islet beta cell under metabolic stress.