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.

Regulatory roles of CARD9-BCL10-Rac1 (CBR) signalome in islet ß-cell function in health and metabolic stress: Is there room for MALT1?

It is widely accepted that pancreatic islet ß-cell failure and the onset of type 2 diabetes (T2DM) constitute an intricate interplay between the genetic expression of the disease and a host of intracellular events including increased metabolic (oxidative, endoplasmic reticulum) stress under the duress of glucolipotoxicity. Emerging evidence implicates unique roles for Caspase Recruitment Domain containing protein 9 (CARD9) in the onset of metabolic diseases, including obesity and insulin resistance. Mechanistically, CARD9 has been implicated in the regulation of p38MAPK and NFkB signaling pathways culminating in cellular dysfunction. Several regulatory factors, including B-cell lymphoma/leukemia 10 (BCL10) have been identified as modulators of CARD9 function in multiple cell types. Despite this evidence on regulatory roles of CARD9-BCL10 signalome in the onset of various pathological states, putative roles of this signaling module in islet ß-cell dysfunction in metabolic stress remain less understood. This brief review is aimed at highlighting roles for CARD9 in islet ß-cell function under acute (physiological insulin secretion) and long-term (cell dysfunction) exposure to glucose. Emerging roles of other signaling proteins, such as Rac1, BCL10 and MALT1 as contributors to CARD9 signaling in the islet ß-cells are also reviewed. Potential avenues for future research toward the development of novel therapeutics for the prevention CARD9-BCL10-Rac1 (CBR) signalome-induced ß-cell defects under metabolic stress are discussed.

Inhibition of GPR39 restores defects in endothelial cell-mediated neovascularization under the duress of chronic hyperglycemia: Evidence for regulatory roles of the sonic hedgehog signaling axis.

Impaired endothelial cell (EC)-mediated angiogenesis contributes to critical limb ischemia in diabetic patients. The sonic hedgehog (SHH) pathway participates in angiogenesis but is repressed in hyperglycemia by obscure mechanisms. We investigated the orphan G protein-coupled receptor GPR39 on SHH pathway activation in ECs and ischemia-induced angiogenesis in animals with chronic hyperglycemia. Human aortic ECs from healthy and type 2 diabetic (T2D) donors were cultured in vitro. GPR39 mRNA expression was significantly elevated in T2D. The EC proliferation, migration, and tube formation were attenuated by adenovirus-mediated GPR39 overexpression (Ad-GPR39) or GPR39 agonist TC-G-1008 in vitro. The production of proangiogenic factors was reduced by Ad-GPR39. Conversely, human ECs transfected with GPR39 siRNA or the mouse aortic ECs isolated from GPR39 global knockout (GPR39KO) mice displayed enhanced migration and proliferation compared with their respective controls. GPR39 suppressed the basal and ligand-dependent activation of the SHH effector GLI1, leading to attenuated EC migration. Coimmunoprecipitation revealed that the GPR39 direct binding of the suppressor of fused (SUFU), the SHH pathway endogenous inhibitor, may achieve this. Furthermore, in ECs with GPR39 knockdown, the robust GLI1 activation and EC migration were abolished by SUFU overexpression. In a chronic diabetic model of diet-induced obesity (DIO) and low-dose streptozotocin (STZ)-induced hyperglycemia, the GPR39KO mice demonstrated a faster pace of revascularization from hind limb ischemia and lower incidence of tissue necrosis than GPR39 wild-type (GPR39WT) counterparts. These findings have provided a conceptual framework for developing therapeutic tools that ablate or inhibit GPR39 for ischemic tissue repair under metabolic stress.

Alpha4 contributes to the dysfunction of the pancreatic beta cell under metabolic stress.

The current study examined the roles of Alpha4, a non-canonical subunit of protein phosphatase 2A, in the regulation of acute (insulin secretion) and chronic (cell dysfunction) effects of glucose in pancreatic beta cells. Alpha4 is expressed in human islets, rat islets and INS-1832/13 cells. Incubation of INS-1832/13 cells and rat islets with high glucose (HG) significantly increased the expression of Alpha4. C2-Ceramide, a biologically active sphingolipid, also increased the expression of Alpha4 in INS-1832/13 cells and rat islets. Subcellular distribution studies of Alpha4 in low glucose (LG) and HG exposed INS-1832/13 cells revealed that it is predominantly cytosolic, and its expression is significantly increased in the non-nuclear/cytosolic fractions in cells exposed to HG. siRNA-mediated knockdown of Alpha4 exerted minimal effects on glucose- or KCl-induced insulin secretion. siRNA-mediated deletion of Alpha4 significantly increased p38MAPK and JNK1/2 phosphorylation under LG conditions, comparable to the degree seen under HG conditions. Paradoxically, a significant potentiation of HG-induced p38MAPK and JNK2 phosphorylation was noted following Alpha4 deletion. HG-induced CHOP expression (ER stress marker) and caspase-3 activation were markedly attenuated in cells following Alpha4 knockdown. Deletion of Alpha4 in INS-1832/13 cells prevented HG-induced loss in the expression of Connexin36, a gap junction channel protein, which has been implicated in normal beta cell function. Lastly, depletion of endogenous Alpha4 significantly reduced HG-induced cell death in INS-1832/13 cells. Based on these findings we conclude that Alpha4 contributes to HG-induced metabolic dysfunction of the islet beta cell.

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.

CARD9 Mediates Pancreatic Islet Beta-Cell Dysfunction Under the Duress of Hyperglycemic Stress.

BACKGROUND/AIMS: Published evidence implicates Caspase recruitment domain containing protein 9 (CARD9) in innate immunity. Given its recently suggested roles in obesity and insulin resistance, we investigated its regulatory role(s) in the onset of islet beta cell dysfunction under chronic hyperglycemic (metabolic stress) conditions. METHODS: Islets from mouse pancreas were isolated by the collagenase digestion method. Expression of CARD9 was suppressed in INS-1 832/13 cells by siRNA transfection using the DharmaFect1 reagent. The degree of activation of Rac1 was assessed by a pull-down assay kit. Interactions between CARD9, RhoGDIß and Rac1 under metabolic stress conditions were determined by co-immunoprecipitation assay. The degree of phosphorylation of stress kinases was assessed using antibodies directed against phosphorylated forms of the respective kinases. RESULTS: CARD9 expression is significantly increased following exposure to high glucose, not to mannitol (both at 20 mM; 24 hrs.) in INS-1 832/13 cells. siRNA-mediated knockdown of CARD9 significantly attenuated high glucose-induced activation of Rac1 and phosphorylation of p38MAPK and p65 subunit of NF-κB (RelA), without significantly impacting high glucose-induced effects on JNK1/2 and ERK1/2 activities. CARD9 depletion also suppressed high glucose-induced CHOP expression (a marker for endoplasmic reticulum stress) in these cells. Co-immunoprecipitation studies revealed increased association between CARD9-RhoGDIß and decreased association between RhoGDIß-Rac1 in cells cultured under high glucose conditions. CONCLUSION: Based on these data, we conclude that CARD9 regulates activation of Rac1-p38MAPK-NFκB signaling pathway leading to functional abnormalities in beta cells under metabolic stress conditions.

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.

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 ß-cell.

Metformin Increases Protein Phosphatase 2A Activity in Primary Human Skeletal Muscle Cells Derived from Lean Healthy Participants.

OBJECTIVE: To investigate if PP2A plays a role in metformin-induced insulin sensitivity improvement in human skeletal muscle cells. Participants. Eight lean insulin-sensitive nondiabetic participants (4 females and 4 males; age: 21.0 ± 1.0 years; BMI: 22.0 ± 0.7 kg/m2; 2-hour OGTT: 97.0 ± 6.0 mg/dl; HbA1c: 5.3 ± 0.1%; fasting plasma glucose: 87.0 ± 2.0 mg/dl; M value; 11.0 ± 1.0 mg/kgBW/min). DESIGN: A hyperinsulinemic-euglycemic clamp was performed to assess insulin sensitivity in human subjects, and skeletal muscle biopsy samples were obtained. Primary human skeletal muscle cells (shown to retain metabolic characteristics of donors) were cultured from these muscle biopsies that included 8 lean insulin-sensitive participants. Cultured cells were expanded, differentiated into myotubes, and treated with 50 µM metformin for 24 hours before harvesting. PP2Ac activity was measured by a phosphatase activity assay kit (Millipore) according to the manufacturer's protocol. RESULTS: The results indicated that metformin significantly increased the activity of PP2A in the myotubes for all 8 lean insulin-sensitive nondiabetic participants, and the average fold increase is 1.54 ± 0.11 (P < 0.001). CONCLUSIONS: These results provided the first evidence that metformin can activate PP2A in human skeletal muscle cells derived from lean healthy insulin-sensitive participants and may help to understand metformin's action in skeletal muscle in humans.

Novel Role of GPR35 (G-Protein-Coupled Receptor 35) in the Regulation of Endothelial Cell Function and Blood Pressure.

[Figure: see text].

CARD9 mediates glucose-stimulated insulin secretion in pancreatic beta cells.

Caspase recruitment domain containing protein 9 (CARD9) plays key regulatory role(s) in innate and adaptive immune responses. Recent evidence implicates CARD9 in the onset of metabolic diseases including insulin resistance. However, potential contributory roles of CARD9 in glucose-stimulated insulin secretion (GSIS) remain unknown. Herein, we report that CARD9 is expressed in human islets, rat islets, mouse islets and clonal INS-1 832/13 cells. Subcellularly, CARD9 is predominantly cytosolic (~75%) in INS-1 832/13 cells. siRNA-mediated depletion of CARD9 expression significantly (~50%) suppressed GSIS in INS-1 832/13 cells. Interestingly, glucose-induced activation of Rac1, a small G-protein, which is a requisite for GSIS to occur, is unaffected in CARD9-si transfected cells, suggesting that CARD9-mediates GSIS in a Rac1-independent fashion. Furthermore, insulin secretion promoted by KCl or mastoparan (a global G protein activator), remained resistant to CARD9 depletion in INS-1 832/13 cells. In addition, pharmacological inhibition (BRD5529) of interaction between CARD9 and TRIM62, its ubiquitin ligase, exerted no significant effects on GSIS. Lastly, depletion of CARD9 prevented glucose-induced p38, not ERK1/2 phosphorylation in beta cells. Based on these observations, we propose that CARD9 might regulate GSIS via a Rac1-independent and p38-dependent signaling module.

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.

Paradoxical regulation of glucose-induced Rac1 activation and insulin secretion by RhoGDIß in pancreatic ß-cells.

Small GTPases (e.g., Rac1) play key roles in glucose-stimulated insulin secretion (GSIS) in the ß-cell. We investigated regulation by RhoGDIß of glucose-induced activation of Rac1 and insulin secretion. RhoGDIß is expressed in INS-1 832/13 cells, rodent and human islets. siRNA-mediated knockdown of RhoGDIß in INS-1 832/13 cells significantly attenuated glucose-induced Rac1 activation without affecting its translocation and membrane association. Further, suppression of RhoGDIß expression exerted minimal effects on GSIS at the height of inhibition of Rac1 activation, suggesting divergent effects of RhoGDIß on Rac1 activation and insulin secretion in the glucose-stimulated ß-cell. We provide the first evidence for the expression of RhoGDIß in rodent and human ß-cells, and its differential regulatory roles of this protein in G protein activation and GSIS. Abbreviations: Arf6: ADP ribosylation factor; Cdc42: Cell Division Cycle; GAP: GTPase-activating protein; GDI: GDP dissociation inhibitor; GDIα: GDP dissociation inhibitorα; GDIß: GDP dissociation inhibitorß; GEF: Guanine nucleotide exchange factor; GSIS: Glucose-stimulated insulin secretion; Rac1: Ras-Related C3 Botulinum Toxin Substrate 1.

Roles of GTP and Rho GTPases in pancreatic islet beta cell function and dysfunction.

A growing body of evidence implicates requisite roles for GTP and its binding proteins (Rho GTPases) in the cascade of events leading to physiological insulin secretion from the islet beta cell. Interestingly, chronic exposure of these cells to hyperglycaemic conditions appears to result in sustained activation of specific Rho GTPases (e.g. Rac1) leading to significant alterations in cellular functions including defects in mitochondrial function and nuclear collapse culminating in beta cell demise. One of the objectives of this review is to highlight our current understanding of the regulatory roles of GTP and Rho GTPases in normal islet function (e.g. proliferation and insulin secretion) as well potential defects in these signalling molecules and metabolic pathways that could contribute islet beta cell dysfunction and loss of functional beta cell mass leading to the onset of diabetes. Potential knowledge gaps in this field and possible avenues for future research are also highlighted.Abbreviations: ARNO: ADP-ribosylation factor nucleotide binding site opener; CML: carboxyl methylation; Epac: exchange protein directly activated by cAMP; ER stress: endoplasmic reticulum stress; FTase: farnesyltransferase; GAP: GTPase activating protein; GDI: GDP dissociation inhibitor; GEF: guanine nucleotide exchange factor; GGTase: geranylgeranyltransferase; GGpp: geranylgeranylpyrophosphate; GGPPS: geranylgeranyl pyrophosphate synthase; GSIS: glucose-stimulated insulin secretion; HGPRTase: hypoxanthine-guanine phosphoribosyltransferase; IMPDH: inosine monophosphate dehydrogenase; α-KIC: α-ketoisocaproic acid; MPA: mycophenolic acid; MVA: mevalonic acid; NDPK: nucleoside diphosphate kinase; NMPK: nucleoside monophosphate kinase; Nox2: phagocyte-like NADPH oxidase; PAK-I: p21-activated kinase-I; ß-PIX: ß-Pak-interacting exchange factor; PRMT: protein arginine methyltransferase; Rac1: ras-related C3 botulinum toxin substrate 1; Tiam1: T-cell lymphoma invasion and metastasis-inducing protein 1; Trx-1: thioredoxin-1; Vav2: vav guanine nucleotide exchange factor 2.

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

BACKGROUND/AIMS: 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 ß-cell. Therefore, we investigated potential roles for P-Rex1 in glucose-stimulated Rac1 activation and insulin secretion in insulin-secreting (INS-1 832/13) ß-cells. METHODS: 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. RESULTS: 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. CONCLUSION: Based on these findings, we conclude that P-Rex1 plays a novel regulatory role in glucose-induced Rac1 activation and insulin secretion.

Oxidative Stress in Cytokine-Induced Dysfunction of the Pancreatic Beta Cell: Known Knowns and Known Unknowns.

Compelling evidence from earlier studies suggests that the pancreatic beta cell is inherently weak in its antioxidant defense mechanisms to face the burden of protecting itself against the increased intracellular oxidative stress following exposure to proinflammatory cytokines. Recent evidence implicates novel roles for nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (Noxs) as contributors to the excessive intracellular oxidative stress and damage under metabolic stress conditions. This review highlights the existing evidence on the regulatory roles of at least three forms of Noxs, namely Nox1, Nox2, and Nox4, in the cascade of events leading to islet beta cell dysfunction, specifically under the duress of chronic exposure to cytokines. Potential crosstalk between key signaling pathways (e.g., inducible nitric oxide synthase [iNOS] and Noxs) in the generation and propagation of reactive molecules and metabolites leading to mitochondrial damage and cell apoptosis is discussed. Available data accrued in investigations involving small-molecule inhibitors and antioxidant protein expression methods as tools toward the prevention of cytokine-induced oxidative damage are reviewed. Lastly, current knowledge gaps in this field, and possible avenues for future research are highlighted.

Potential roles of PP2A-Rac1 signaling axis in pancreatic ß-cell dysfunction under metabolic stress: Progress and promise.

Recent estimates by the International Diabetes Federation suggest that the incidence of diabetes soared to an all-time high of 463 million in 2019, and the federation predicts that by 2045 the number of individuals afflicted with this disease will increase to 700 million. Therefore, efforts to understand the pathophysiology of diabetes are critical for moving toward the development of novel therapeutic strategies for this disease. Several contributors (oxidative stress, endoplasmic reticulum stress and others) have been proposed for the onset of metabolic dysfunction and demise of the islet ß-cell leading to the pathogenesis of diabetes. Existing experimental evidence revealed sustained activation of PP2A and Rac1 in pancreatic ß-cells exposed to metabolic stress (diabetogenic) conditions. Evidence in a variety of cell types implicates modulatory roles for specific signaling proteins (α4, SET, nm23-H1, Pak1) in the functional regulation of PP2A and Rac1. In this Commentary, I overviewed potential cross-talk between PP2A and Rac1 signaling modules in the onset of metabolic dysregulation of the islet ß-cell leading to impaired glucose-stimulated insulin secretion (GSIS), loss of ß-cell mass and the onset of diabetes. Potential knowledge gaps and future directions in this fertile area of islet biology are also highlighted. It is hoped that this Commentary will provide a basis for future studies toward a better understanding of roles of PP2A-Rac1 signaling module in pancreatic ß-cell dysfunction, and identification of therapeutic targets for the treatment of islet ß-cell dysfunction in diabetes.

GPCRs, G Proteins, and Their Impact on ß-cell Function.

Glucose-induced (physiological) insulin secretion from the islet ß-cell involves interplay between cationic (i.e., changes in intracellular calcium) and metabolic (i.e., generation of hydrophobic and hydrophilic second messengers) events. A large body of evidence affirms support for novel regulation, by G proteins, of specific intracellular signaling events, including actin cytoskeletal remodeling, transport of insulin-containing granules to the plasma membrane for fusion, and secretion of insulin into the circulation. This article highlights the following aspects of GPCR-G protein biology of the islet. First, it overviews our current understanding of the identity of a wide variety of G protein regulators and their modulatory roles in GPCR-G protein-effector coupling, which is requisite for optimal ß-cell function under physiological conditions. Second, it describes evidence in support of novel, noncanonical, GPCR-independent mechanisms of activation of G proteins in the islet. Third, it highlights the evidence indicating that abnormalities in G protein function lead to islet ß-cell dysregulation and demise under the duress of metabolic stress and diabetes. Fourth, it summarizes observations of potential beneficial effects of GPCR agonists in preventing/halting metabolic defects in the islet ß-cell under various pathological conditions (e.g., metabolic stress and inflammation). Lastly, it identifies knowledge gaps and potential avenues for future research in this evolving field of translational islet biology. Published 2020. Compr Physiol 10:453-490, 2020.