Publications by authors named "Anjaneyulu Kowluru"

92 Publications

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

Cell Physiol Biochem 2021 Apr;55(2):180-192

Biomedical Research Service, John D. Dingell VA Medical Center, Detroit, MI, USA,

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 ELISA 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.
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http://dx.doi.org/10.33594/000000354DOI Listing
April 2021

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

Cell Physiol Biochem 2020 Dec;54(6):1218-1230

Biomedical Research Service, John D. Dingell VA Medical Center, Detroit, MI, USA,

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 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.

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.
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http://dx.doi.org/10.33594/000000310DOI Listing
December 2020

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

Metabolites 2020 Nov 24;10(12). Epub 2020 Nov 24.

Biomedical Research Service, John D. Dingell VA Medical Center, Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, Detroit, MI 48201, USA.

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.
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http://dx.doi.org/10.3390/metabo10120480DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7759861PMC
November 2020

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

Small GTPases 2020 Aug 31:1-13. Epub 2020 Aug 31.

Biomedical Research Service, John D. Dingell VA Medical Center and Department of Pharmaceutical Sciences and Medicine, Wayne State University, Detroit, MI, USA.

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; β-PIX: β-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.
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http://dx.doi.org/10.1080/21541248.2020.1815508DOI Listing
August 2020

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

Biochem Pharmacol 2020 10 4;180:114138. Epub 2020 Jul 4.

Biomedical Laboratory Research Service, John D. Dingell VA Medical Center and Departments of Pharmaceutical Sciences and Internal Medicine, Wayne State University, Detroit, MI 48201, USA. Electronic address:

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 β-cell leading to the pathogenesis of diabetes. Existing experimental evidence revealed sustained activation of PP2A and Rac1 in pancreatic β-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 β-cell leading to impaired glucose-stimulated insulin secretion (GSIS), loss of β-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 β-cell dysfunction, and identification of therapeutic targets for the treatment of islet β-cell dysfunction in diabetes.
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http://dx.doi.org/10.1016/j.bcp.2020.114138DOI Listing
October 2020

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

Compr Physiol 2020 03 12;10(2):453-490. Epub 2020 Mar 12.

Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Center for Translational Research in Diabetes, Biomedical Research Service, John D. Dingell VA Medical Center, Wayne State University, Detroit, Michigan, USA.

Glucose-induced (physiological) insulin secretion from the islet β-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 β-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 β-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 β-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.
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http://dx.doi.org/10.1002/cphy.c190028DOI Listing
March 2020

Kinome Profiling Reveals Abnormal Activity of Kinases in Skeletal Muscle From Adults With Obesity and Insulin Resistance.

J Clin Endocrinol Metab 2020 Mar;105(3)

Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, Wayne State University, Detroit, MI.

Context: Obesity-related insulin resistance (OIR) is one of the main contributors to type 2 diabetes and other metabolic diseases. Protein kinases are implicated in insulin signaling and glucose metabolism. Molecular mechanisms underlying OIR involving global kinase activities remain incompletely understood.

Objective: To investigate abnormal kinase activity associated with OIR in human skeletal muscle.

Design: Utilization of stable isotopic labeling-based quantitative proteomics combined with affinity-based active enzyme probes to profile in vivo kinase activity in skeletal muscle from lean control (Lean) and OIR participants.

Participants: A total of 16 nondiabetic adults, 8 Lean and 8 with OIR, underwent hyperinsulinemic-euglycemic clamp with muscle biopsy.

Results: We identified the first active kinome, comprising 54 active protein kinases, in human skeletal muscle. The activities of 23 kinases were different in OIR muscle compared with Lean muscle (11 hyper- and 12 hypo-active), while their protein abundance was the same between the 2 groups. The activities of multiple kinases involved in adenosine monophosphate-activated protein kinase (AMPK) and p38 signaling were lower in OIR compared with Lean. On the contrary, multiple kinases in the c-Jun N-terminal kinase (JNK) signaling pathway exhibited higher activity in OIR vs Lean. The kinase-substrate-prediction based on experimental data further confirmed a potential downregulation of insulin signaling (eg, inhibited phosphorylation of insulin receptor substrate-1 and AKT1/2).

Conclusions: These findings provide a global view of the kinome activity in OIR and Lean muscle, pinpoint novel specific impairment in kinase activities in signaling pathways important for skeletal muscle insulin resistance, and may provide potential drug targets (ie, abnormal kinase activities) to prevent and/or reverse skeletal muscle insulin resistance in humans.
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http://dx.doi.org/10.1210/clinem/dgz115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6991621PMC
March 2020

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

Small GTPases 2021 Mar 3;12(2):114-121. Epub 2019 Jul 3.

Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University , Detroit, USA.

Small GTPases (e.g., Rac1) play key roles in glucose-stimulated insulin secretion (GSIS) in the β-cell. We investigated regulation by RhoGDIβ of glucose-induced activation of Rac1 and insulin secretion. RhoGDIβ is expressed in INS-1 832/13 cells, rodent and human islets. siRNA-mediated knockdown of RhoGDIβ in INS-1 832/13 cells significantly attenuated glucose-induced Rac1 activation without affecting its translocation and membrane association. Further, suppression of RhoGDIβ expression exerted minimal effects on GSIS at the height of inhibition of Rac1 activation, suggesting divergent effects of RhoGDIβ on Rac1 activation and insulin secretion in the glucose-stimulated β-cell. We provide the first evidence for the expression of RhoGDIβ in rodent and human β-cells, and its differential regulatory roles of this protein in G protein activation and GSIS. : Arf6: ADP ribosylation factor; Cdc42: Cell Division Cycle; GAP: GTPase-activating protein; GDI: GDP dissociation inhibitor; GDIα: GDP dissociation inhibitorα; GDIβ: GDP dissociation inhibitorβ; GEF: Guanine nucleotide exchange factor; GSIS: Glucose-stimulated insulin secretion; Rac1: Ras-Related C3 Botulinum Toxin Substrate 1.
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http://dx.doi.org/10.1080/21541248.2019.1635403DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7849774PMC
March 2021

Quantitative proteomics reveals novel interaction partners of Rac1 in pancreatic β-cells: Evidence for increased interaction with Rac1 under hyperglycemic conditions.

Mol Cell Endocrinol 2019 08 13;494:110489. Epub 2019 Jun 13.

Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, USA; Center for Translational Research in Diabetes, Biomedical Research Service, John D. Dingell VA Medical Center, Detroit, MI, 48201, USA. Electronic address:

Rac1, a small G protein, regulates physiological insulin secretion from the pancreatic β-cell. Interestingly, Rac1 has also been implicated in the onset of metabolic dysfunction of the β-cell under the duress of hyperglycemia (HG). This study is aimed at the identification of interaction partners of Rac1 in β-cells under basal and HG conditions. Using co-immunoprecipitation and UPLC-ESI-MS/MS, we identified 324 Rac1 interaction partners in INS-1832/13 cells, which represent the largest Rac1 interactome to date. Furthermore, we identified 27 interaction partners that exhibited increased association with Rac1 in β-cells exposed to HG. Western blotting (INS-1832/13 cells, rat islets and human islets) and co-immunoprecipitation (INS-1832/13 cells) further validated the identity of these Rac1 interaction partners including regulators of GPCR-G protein-effector coupling in the islet. These data form the basis for future investigations on contributory roles of these Rac1-specific signaling pathways in islet β-cell function in health and diabetes.
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http://dx.doi.org/10.1016/j.mce.2019.110489DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6686664PMC
August 2019

Epigenetics and Regulation of Oxidative Stress in Diabetic Retinopathy.

Invest Ophthalmol Vis Sci 2018 10;59(12):4831-4840

Department of Ophthalmology, Wayne State University, Detroit, Michigan, United States.

Purpose: Oxidative stress plays a central role in the development of diabetic retinopathy, and in the pathogenesis of this blinding disease, activation of NADPH oxidase 2 (Nox2)-mediated cytosolic reactive oxygen species (ROS) production precedes mitochondrial damage. The multicomponent cytosolic Nox2 has an obligatory component, Ras-related C3 botulinum toxin substrate 1 (Rac1); in diabetes, Rac1 is functionally and transcriptionally active. Diabetes also facilitates many epigenetic modifications, and activates both DNA methylating (Dnmts) and hydroxymethylating (Tets) enzymes. Our aim was to investigate the role of epigenetics in Rac1 regulation in diabetes.

Methods: Using human retinal endothelial cells, exposed to high glucose, 5-methyl cytosine (5mC) and 5-hydroxy methyl cytosine (5hmC) levels, and binding of Dnmt and Tets were quantified at the Rac1 promoter. The effect of inhibition of Dnmts/Tets (pharmacological inhibitors or short interfering RNA [siRNA]) on glucose-induced activation of Rac1-ROS production was evaluated. Results were confirmed in retinal microvessels from streptozotocin-induced diabetic mice receiving intravitreally Dnmt1-siRNA.

Results: Despite high glucose-induced increased binding of Dnmt1, 5mC levels remained subnormal at Rac1 promoter. But, at the same site, 5hmC levels and transcription factor nuclear factor (NF)-kB binding were increased. Inhibition of Dnmts/Tets prevented increase in 5hmC and NF-kB binding, and attenuated Rac1 activation. Similarly, in mouse retinal microvessels, Dnmt1-siRNA ameliorated diabetes-induced increase in Rac1 transcripts and activity, and decreased ROS levels.

Conclusions: Thus, despite Dnmts activation, concomitant increase in Tets rapidly hydroxymethylates 5mC, allowing NF-κB to bind and activate Rac1. These results imply a critical role of an active DNA methylation in cytosolic ROS regulation in the development of diabetic retinopathy.
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http://dx.doi.org/10.1167/iovs.18-24548DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6181189PMC
October 2018

RACking up ceramide-induced islet β-cell dysfunction.

Biochem Pharmacol 2018 08 30;154:161-169. Epub 2018 Apr 30.

Department of Ophthalmology and Anatomy and Cell Biology, Wayne State University, Detroit, MI 48201, USA.

The International Diabetes Federation predicts that by 2045 the number of individuals afflicted with diabetes will increase to 629 million. Furthermore, ∼352 million individuals with impaired glucose tolerance are at increased risk for developing diabetes. Several mechanisms have been proposed for the onset of metabolic dysfunction and demise of the islet β-cell leading to the pathogenesis of diabetes. It is widely accepted that the onset of type 2 diabetes is due to an intricate interplay between genetic expression of the disease and a multitude of factors including increased oxidative and endoplasmic reticulum stress consequential to glucolipotoxicity and inflammation. Compelling experimental evidence from in vitro and in vivo studies implicates intracellular generation of ceramide (CER), a biologically-active sphingolipid, as a trigger in the onset of β-cell demise under above pathological conditions. Recent pharmacological and molecular biological evidence affirms regulatory roles for Ras-related C3 botulinum toxin substrate 1 (Rac1), a small G protein, in the islet β-cell function in health and diabetes. In this Commentary, we overviewed the emerging evidence implicating potential cross-talk between Rac1 and ceramide signaling pathways in the onset of metabolic dysregulation of the islet β-cell culminating in impaired physiological insulin secretion, loss of β-cell mass and the onset of diabetes. Further, we propose a model depicting contributory roles of defective protein lipidation (prenylation) pathway in the induction of metabolic defects in the β-cell under metabolic stress conditions. Potential avenues for the identification of novel therapeutic targets for the prevention/treatment of diabetes and its associated complications are highlighted.
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http://dx.doi.org/10.1016/j.bcp.2018.04.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6051906PMC
August 2018

CD36 mediates lipid accumulation in pancreatic beta cells under the duress of glucolipotoxic conditions: Novel roles of lysine deacetylases.

Biochem Biophys Res Commun 2018 01 20;495(3):2221-2226. Epub 2017 Dec 20.

β-Cell Biochemistry Laboratory, John D. Dingell VA Medical Center, and Department of Pharmaceutical Sciences, Wayne State University, Detroit, MI, 48201, USA. Electronic address:

The cluster of differentiation 36 (CD36) is implicated in the intake of long-chain fatty acids and fat storage in various cell types including the pancreatic beta cell, thus contributing to the pathogenesis of metabolic stress and diabetes. Recent evidence indicates that CD36 undergoes post-translational modifications such as acetylation-deacetylation. However, putative roles of such modifications in its functional activation and onset of beta cell dysregulation under the duress of glucolipotoxicity (GLT) remain largely unknown. Using pharmacological approaches, we validated, herein, the hypothesis that acetylation-deacetylation signaling steps are involved in CD36-mediated lipid accumulation and downstream apoptotic signaling in pancreatic beta (INS-1832/13) cells under GLT. Exposure of these cells to GLT resulted in significant lipid accumulation without affecting the CD36 expression. Sulfo-n-succinimidyl oleate (SSO), an irreversible inhibitor of CD36, significantly attenuated lipid accumulation under GLT conditions, thus implicating CD36 in this metabolic step. Furthermore, trichostatin A (TSA) or valproic acid (VPA), known inhibitors of lysine deacetylases, markedly suppressed GLT-associated lipid accumulation with no discernible effects on CD36 expression. Lastly, SSO or TSA prevented caspase 3 activation in INS-1832/13 cells exposed to GLT conditions. Based on these findings, we conclude that an acetylation-deacetylation signaling step might regulate CD36 functional activity and subsequent lipid accumulation and caspase 3 activation in pancreatic beta cells exposed to GLT conditions. Identification of specific lysine deacetylases that control CD36 function should provide novel clues for the prevention of beta-cell dysfunction under GLT.
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http://dx.doi.org/10.1016/j.bbrc.2017.12.111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5756509PMC
January 2018

Role of G-proteins in islet function in health and diabetes.

Diabetes Obes Metab 2017 09;19 Suppl 1:63-75

β-Cell Biochemistry Laboratory, John D. Dingell VA Medical Center, and Department of Pharmaceutical Sciences, Wayne State University, Detroit, Michigan.

Glucose-stimulated insulin secretion (GSIS) involves interplay between metabolic and cationic events. Seminal contributions from multiple laboratories affirm essential roles for small G-proteins (Rac1, Cdc42, Arf6, Rab27A) in GSIS. Activation of these signalling proteins promotes cytoskeletal remodeling, transport and docking of insulin granules on the plasma membrane for exocytotic secretion of insulin. Evidence in rodent and human islets suggests key roles for lipidation (farnesylation and geranylgeranylation) of these G-proteins for their targeting to appropriate cellular compartments for optimal regulation of effectors leading to GSIS. Interestingly, however, inhibition of prenylation appears to cause mislocalization of non-prenylated, but (paradoxically) activated G-proteins, in "inappropriate" compartments leading to activation of stress kinases and onset of mitochondrial defects, loss in GSIS and apoptosis of the islet β-cell. This review highlights our current understanding of roles of G-proteins and their post-translational lipidation (prenylation) signalling networks in islet function in normal health, metabolic stress (glucolipotoxicity and ER stress) and diabetes. Critical knowledge gaps that need to be addressed for the development of therapeutics to halt defects in these signalling steps in β-cells in models of impaired insulin secretion and diabetes are also highlighted and discussed.
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http://dx.doi.org/10.1111/dom.13011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5657296PMC
September 2017

Glucotoxicity promotes aberrant activation and mislocalization of Ras-related C3 botulinum toxin substrate 1 [Rac1] and metabolic dysfunction in pancreatic islet β-cells: reversal of such metabolic defects by metformin.

Apoptosis 2017 Nov;22(11):1380-1393

β-Cell Biochemistry Laboratory, John D. Dingell VA Medical Center, Detroit, MI, USA.

Emerging evidence suggests that long-term exposure of insulin-secreting pancreatic β-cells to hyperglycemic (HG; glucotoxic) conditions promotes oxidative stress, which, in turn, leads to stress kinase activation, mitochondrial dysfunction, loss of nuclear structure and integrity and cell apoptosis. Original observations from our laboratory have proposed that Rac1 plays a key regulatory role in the generation of oxidative stress and downstream signaling events culminating in the onset of dysfunction of pancreatic β-cells under the duress of metabolic stress. However, precise molecular and cellular mechanisms underlying the metabolic roles of hyperactive Rac1 remain less understood. Using pharmacological and molecular biological approaches, we now report mistargetting of biologically-active Rac1 [GTP-bound conformation] to the nuclear compartment in clonal INS-1 cells, normal rat islets and human islets under HG conditions. Our findings also suggest that such a signaling step is independent of post-translational prenylation of Rac1. Evidence is also presented to highlight novel roles for sustained activation of Rac1 in HG-induced expression of Cluster of Differentiation 36 [CD36], a fatty acid transporter protein, which is implicated in cell apoptosis. Finally, our findings suggest that metformin, a biguanide anti-diabetic drug, at a clinically relevant concentration, prevents β-cell defects [Rac1 activation, nuclear association, CD36 expression, stress kinase and caspase-3 activation, and loss in metabolic viability] under the duress of glucotoxicity. Potential implications of these findings in the context of novel and direct regulation of islet β-cell function by metformin are discussed.
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http://dx.doi.org/10.1007/s10495-017-1409-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5643241PMC
November 2017

Inappropriate movement of Rac1 contributes to glucotoxicity of the islet β-cell.

Cell Cycle 2017 08 19;16(15):1387-1388. Epub 2017 Jul 19.

a β-Cell Biochemistry Laboratory, John D. Dingell VA Medical Center, Department of Pharmaceutical Sciences , Wayne State University , Detroit , MI , USA.

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http://dx.doi.org/10.1080/15384101.2017.1345229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5553397PMC
August 2017

Exposure to chronic hyperglycemic conditions results in Ras-related C3 botulinum toxin substrate 1 (Rac1)-mediated activation of p53 and ATM kinase in pancreatic β-cells.

Apoptosis 2017 05;22(5):597-607

Beta-Cell Biochemistry Laboratory, John D. Dingell VA Medical Center, B-4237 Research Service, 4646 John R, Detroit, MI, 48201, USA.

Chronic hyperglycemia (HG) promotes pancreatic islet dysfunction which leads to the onset of T2DM. This study is aimed at defining regulatory roles of Rac1, a small G-protein, in the activation of p53 and ATM kinase in pancreatic β-cells, under the duress of HG conditions. We report significant stimulatory effects of HG (20 mM; 24 h) on p53 activation in INS-1 832/13 cells, normal rodent and human islets. Pharmacological inhibition of Rac1 (EHT1864 or NSC23766) significantly suppressed HG-induced p53 activation in INS-1 832/13 cells and rat islets, suggesting novel roles for this small G-protein in the activation of p53. Inhibition of Rac1 geranylgeranylation with simvastatin or GGTI-2147, significantly attenuated HG-induced p53 activation, suggesting requisite roles for this signaling step in HG-mediated effects on β-cells. HG-induced p53 activation was also suppressed by SB203580, a known inhibitor of p38MAPK. Additionally, we observed increased activation of ATM kinase under HG conditions, which was blocked in presence of EHT1864. Furthermore, pharmacological inhibition of ATM kinase (KU55933) reduced activation of ATM kinase, but not p53, suggesting that HG-mediated activation of p53 and ATM could represent independent pro-apoptotic events. In conclusion, these data indicate that sustained activation of Rac1-p38MAPK signaling axis leads to activation of p53 leading to β-cell dysfunction under the duress of chronic hyperglycemic conditions.
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http://dx.doi.org/10.1007/s10495-017-1354-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6685530PMC
May 2017

Tiam1/Vav2-Rac1 axis: A tug-of-war between islet function and dysfunction.

Biochem Pharmacol 2017 05 13;132:9-17. Epub 2017 Feb 13.

Beta-Cell Biochemistry Laboratory, John D. Dingell VA Medical Center, and Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, Detroit, MI 48201, USA. Electronic address:

Glucose-stimulated insulin secretion [GSIS] from the islet β-cell involves a well-orchestrated interplay between metabolic and cationic events. It is well established that intracellular generation of adenine and guanine nucleotide triphosphates [e.g., ATP and GTP] represents one of the requisite signaling steps in GSIS. The small molecular mass GTP-binding proteins [G-proteins; e.g., Rac1 and Cdc42] have been shown to regulate islet β-cell function including actin cytoskeletal remodeling and fusion of insulin granules with the plasma membrane for GSIS to occur. In this context, several regulatory factors for these G-proteins have been identified in the pancreatic β-cell; these include guanine nucleotide exchange factors [GEFs] and guanine nucleotide dissociation inhibitors [GDI]. Recent pharmacological and molecular biological evidence identified Tiam1 and Vav2 as GEFs for Rac1 in promoting physiological insulin secretion. Paradoxically, emerging evidence in multiple cell types, including the islet β-cell, suggests key roles for Rac1 in the onset of cellular dysfunction under conditions of metabolic stress and diabetes. Furthermore, functional inactivation of either Tiam1 or Vav2 appears to attenuate sustained activation of Rac1 and its downstream signaling events [activation of stress kinases] under conditions of metabolic stress. Together, these findings suggest both "friendly" and "non-friendly" roles for Tiam1/Vav2-Rac1 signaling axis in islet β-cell in health and diabetes. Our current understanding of the field and the knowledge gaps that exist in this area of islet biology are heighted herein. Furthermore, potential caveats in the specificity and selectivity of pharmacological inhibitors that are available currently are discussed in this Commentary.
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http://dx.doi.org/10.1016/j.bcp.2017.02.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5382093PMC
May 2017

Metabolic Stress Induces Caspase-3 Mediated Degradation and Inactivation of Farnesyl and Geranylgeranyl Transferase Activities in Pancreatic β-Cells.

Cell Physiol Biochem 2016 2;39(6):2110-2120. Epub 2016 Nov 2.

Department of Molecular and Cellular Endocrinology, Diabetes and Metabolic Research Institute, City of Hope/BRI, Duarte, USA.

Background/aims: At least 300 prenylated proteins are identified in the human genome; the majority of which partake in a variety of cellular processes including growth, differentiation, cytoskeletal organization/dynamics and vesicle trafficking. Aberrant prenylation of proteins is implicated in human pathologies including cancer; neurodegenerative diseases, retinitis pigmentosa, and premature ageing syndromes. Original observations from our laboratory have demonstrated that prenylation of proteins [small G-proteins and γ-subunits of trimeric G-proteins] is requisite for physiological insulin secretion. Herein, we assessed the impact of metabolic stress [gluco-, lipotoxicity and ER-stress] on the functional status of protein prenylation pathway in pancreatic β-cells.

Methods: Farnesyltransferase [FTase] and geranylgeranyltransferase [GGTase] activities were quantified by radioisotopic methods. Caspase-3 activation and FTase/GGTase-α subunit degradation were determined by Western blotting.

Results: We observed that metabolic stress activates caspase-3 and induces degradation of the common α-subunit of FTase and GGTase-I in INS-1 832/13 cells, normal rodent islets and human islets leading to functional defects [inactivation] in FTase and GGTase activities. Caspase-3 activation and FTase/GGTase-α degradation were also seen in islets from the Zucker diabetic fatty [ZDF] rat, a model for Type 2 diabetes. Consequential to defects in FTase/GGTase-α signaling, we observed significant accumulation of unprenylated proteins [Rap1] in β-cells exposed to glucotoxic conditions. These findings were replicated in β-cells following pharmacological inhibition of generation of prenylpyrophosphate substrates [Simvastatin] or catalytic activity of prenylating enzymes [GGTI-2147].

Conclusions: Our findings provide the first evidence to suggest that metabolic stress induced dysfunction of the islet β-cell may, in part, be due to defective protein prenylation signaling pathway.
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http://dx.doi.org/10.1159/000447907DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6685439PMC
February 2017

The Regulatory Roles of Mitogen-Activated Protein Kinase (MAPK) Pathways in Health and Diabetes: Lessons Learned from the Pancreatic β-Cell.

Recent Pat Endocr Metab Immune Drug Discov 2017 ;10(2):76-84

B-4237 Research Service, John D. Dingell VA Medical Center, 4646 John R, Detroit, MI 48201. United States.

Background: Glucose-stimulated insulin secretion (GSIS) from the pancreatic β-cell involves several intracellular metabolic events which lead to the translocation of insulin granules towards the membrane for fusion and release. It is well established that loss of β-cell function and decreased GSIS underlie the pathogenesis of diabetes. Evidence from several laboratories, including our own, demonstrated requisite roles of Rac1 and phagocyte-like NADPH oxidase (Nox2)-derived reactive oxygen species (ROS) in optimal function of the pancreatic β-cell, including GSIS. However, it is becoming increasingly clear that prolonged exposure of β-cells to hyperglycemic conditions, leads to sustained activation of Rac1-Nox2 signaling axis culminating in excessive generation of intracellular ROS (oxidative stress) and β-cell dysregulation and demise. Such "cytotoxic" effects of ROS appear to be mediated via the stress-activated protein kinases/mitogen-activated protein kinases (SAPK/MAPK) signaling pathways.

Objective: This review discusses our current understanding of regulation and functions of the conventional MAPKs, namely, ERK1/2, JNK1/2 and p38MAPK.

Conclusion: The MAPK pathways are activated in the presence of various stress stimuli including intracellular ROS, via distinct signaling cascades. Once activated, MAPKs participate in specific intracellular signaling processes via interaction with several downstream kinases including the MAPKactivated protein kinases (MAPKAPKs) and transcription factors including c-jun and p53. We have provided an overview of existing evidence in the islet β-cell on the regulatory roles of these MAPKs in mediating cellular responses to alterations in intracellularly generated ROS, which is mediated by the Rac1-Nox2 signaling module. Additionally, we enlisted recent patents developed to improve β-cell function in diabetes and novel pharmacological agents that target oxidative stress and MAPK pathways.
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http://dx.doi.org/10.2174/1872214810666161020154905DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6685434PMC
November 2017

Hyperlipidemia and the development of diabetic retinopathy: Comparison between type 1 and type 2 animal models.

Metabolism 2016 Oct 30;65(10):1570-81. Epub 2016 Jul 30.

Ophthalmology, Wayne State University, Detroit, MI, United States.

Aim: In the pathogenesis of diabetic retinopathy, reactive oxygen species (ROS) are elevated in the retina and the mitochondria are damaged, resulting in accelerated apoptosis. Dyslipidemia is also considered as one of the major factors in its development, and our aim is to investigate the compounding effect of hyperlipidemia in retinopathy.

Methods: Retinal ROS, mitochondrial damage and vascular pathology were investigated in Zucker diabetic fatty rats (ZDF, type 2 diabetes model), during the age that spans from hyperlipidemia/pre-hyperglycemia (6weeks), to severe hyperglycemia/moderate hyperlipidemia (~12weeks), and ultimately to severe hyperglycemia/hyperlipidemia (20-40weeks). For comparison, retina from streptozotocin-induced Wistar rats (type 1 diabetic for 10-40weeks) was analyzed.

Results: Compared to age-matched lean rats, despite increased retinal cytosolic ROS in 6-week-old ZDF rats, mitochondrial dysfunction and DNA damage were not detected, and in 12-week-old ZDF rats, retinal mitochondria were dysfunctional, but mtDNA damage and vascular pathology (cell apoptosis and degenerative capillaries) were not detectable. Retina from 20-week-old ZDF rats (hyperglycemic for 14weeks or less) had significant mitochondrial dysfunction, mtDNA damage and vascular pathology, and similar abnormalities were observed in 40-week-old ZDF rats. Although retinal mitochondrial dysfunction was observed in Wistar rats diabetic for 20weeks, mtDNA damage and vascular pathology were not detectable till the duration of diabetes was further extended.

Conclusions: Hyperlipidemia, in a hyperglycemic milieu, potentiates mitochondrial damage and augments the development of retinopathy. Control of dyslipidemia in pre-diabetic patients may prevent/delay the development and the progression of this devastating disease.
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http://dx.doi.org/10.1016/j.metabol.2016.07.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5023070PMC
October 2016

NSC23766, a Known Inhibitor of Tiam1-Rac1 Signaling Module, Prevents the Onset of Type 1 Diabetes in the NOD Mouse Model.

Cell Physiol Biochem 2016 29;39(2):760-7. Epub 2016 Jul 29.

Department of Molecular and Cellular Endocrinology, Diabetes and Metabolic Research Institute, City of Hope/BRI, Duarte, USA.

Background/aims: Type 1 diabetes (T1D) is characterized by absolute insulin deficiency due to destruction of pancreatic β-cells by cytokines (e.g., interleukin-1β; IL-1β) released by invading immune cells. The mechanisms by which these cytokines induce β-cell dysfunction remain poorly understood. Recent evidence suggests that excessive generation of reactive oxygen species (ROS) by the phagocyte-like NADPH oxidase2 (Nox2), along with significantly low levels of antioxidants in β-cells, drive them toward oxidative damage. Rac1, a small G-protein, is one of the members of Nox2 holoenzyme. We recently reported that NSC23766, a known inhibitor of Rac1, significantly attenuated cytokine-induced Nox2 activation and ROS generation in pancreatic islet β-cells in vitro. Herein, we determined the effects of NSC23766 (2.5 mg/kg/day, i.p/daily) on the development of diabetes in the NOD mouse, a model for T1D.

Methods: Two groups of experimental animals (Balb/c and NOD mice) received NSC23766, while the two control groups received equal volume of saline. Body weights and blood glucose were measured every week for 34 weeks. Rac1 activation in pancreatic islets was measured by GLISA activation assay. Rac1 and CHOP expression was determined by Western Blotting.

Results: Our findings indicate that administration of NSC23766 significantly prevented the development of spontaneous diabetes in the NOD mice. Furthermore, NSC23766 markedly suppressed Rac1 expression and activity and the endoplasmic reticulum stress (CHOP expression) in NOD islets.

Conclusions: Our findings provide the first evidence implicating the role of Tiam1-Rac1-Nox2 signaling pathway in the onset of spontaneous diabetes in the NOD mouse model.
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http://dx.doi.org/10.1159/000445666DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6686134PMC
February 2017

Quantitative proteomics reveals novel protein interaction partners of PP2A catalytic subunit in pancreatic β-cells.

Mol Cell Endocrinol 2016 Mar 9;424:1-11. Epub 2016 Jan 9.

Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, Detroit, MI, 48201, USA. Electronic address:

Protein phosphatase 2A (PP2A) is one of the major serine/threonine phosphatases. We hypothesize that PP2A regulates signaling cascades in pancreatic β-cells in the context of glucose-stimulated insulin secretion (GSIS). Using co-immunoprecipitation (co-IP) and tandem mass spectrometry, we globally identified the protein interaction partners of the PP2A catalytic subunit (PP2Ac) in insulin-secreting pancreatic β-cells. Among the 514 identified PP2Ac interaction partners, 476 were novel. This represents the first global view of PP2Ac protein-protein interactions caused by hyperglycemic conditions. Additionally, numerous PP2Ac partners were found involved in a variety of signaling pathways in the β-cell function, such as insulin secretion. Our data suggest that PP2A interacts with various signaling proteins necessary for physiological insulin secretion as well as signaling proteins known to regulate cell dysfunction and apoptosis in the pancreatic β-cells.
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http://dx.doi.org/10.1016/j.mce.2016.01.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4779412PMC
March 2016

A lack of 'glue' misplaces Rab27A to cause islet dysfunction in diabetes.

J Pathol 2016 Feb 4;238(3):375-7. Epub 2016 Jan 4.

Research Service, John D Dingell VA Medical Center and Department of Pharmaceutical Sciences, Wayne State University, Detroit, MI, USA.

Glucose-stimulated insulin secretion (GSIS) involves interplay between metabolic and cationic events. Several lines of evidence suggest novel regulatory roles for small G proteins (Rac1, Cdc42, Rab27A) in cytoskeletal remodelling and docking of insulin granules on the plasma membrane for insulin secretion. Emerging evidence implicates novel roles for post-translational prenylation (farnesylation and geranylgeranylation) of G proteins for their targeting to appropriate membranous compartments. While several recent studies were focused on prenylating enzymes in the islet β-cell, a significant knowledge gap exists on the regulatory roles and function of enzymes that mediate intracellular generation of prenyl pyrophosphate substrates (farnesyl and geranylgeranyl pyrophosphates) for prenyltransferases. Recent work published in The Journal of Pathology by Jiang and associates highlights requisite roles for geranylgeranyl pyrophosphate synthase (GGPPS) in islet β-cell function in health and diabetes. These studies are timely and will form the basis for a series of new investigations to further validate roles for G-protein prenylation in GSIS under physiological conditions. They also pave the path towards the identification of potential defects in these signalling pathways in β-cell models of impaired insulin secretion including metabolic stress and diabetes.
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http://dx.doi.org/10.1002/path.4671DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4724292PMC
February 2016

Biology and Regulatory Roles of Nuclear Lamins in Cellular Function and Dysfunction.

Recent Pat Endocr Metab Immune Drug Discov 2015 ;9(2):111-20

B4237, Research Service, John D. Dingell VA Medical Center, 4646 John R, Detroit, MI 48201; USA.

Nuclear lamins, namely lamins A, B and C, surround the nucleoplasmic contents in a meshlike network called the nuclear lamina. These intermediate filaments provide a structural framework to the nuclear envelope (NE), play a role in arrangement of the chromatin within the nucleus, in DNA replication and also participate in DNA damage repair. In order for lamins to be involved in these important nuclear processes and to be functionally active, they undergo a series of post-translational modifications (farnesylation, endoproteolytic cleavage, carboxylmethylation etc.), of which farnesylation is the most studied. Improper farnesylation of lamin proteins, especially lamin A, leads to a number of diseases affecting the striated muscle (e.g. Emery- Dreifuss Muscular Dystrophy, Dilated Cardiomyopathy), adipose tissue (e.g. Dunnigan-type familial partial lipodystrophy) and could result in abnormal senescence and growth deformities (e.g. Progeria syndrome); these are referred to as laminopathies. Despite the existing literature and evidence regarding functions of lamins and diseases associated with abnormal lamin processing, a lot remains to be understood in regards to lamin biology and their role as potential therapeutic targets. In this brief review, we have attempted to summarize the roles of lamins in physiology and pathology of the cell and in type 2 diabetes mellitus [T2DM] and also enlisted patents on methods, systems and devices developed for improving pancreatic beta cell function in diabetes mellitus.
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http://dx.doi.org/10.2174/1872214809666151009120402DOI Listing
October 2016

VAV2, a guanine nucleotide exchange factor for Rac1, regulates glucose-stimulated insulin secretion in pancreatic beta cells.

Diabetologia 2015 Nov 31;58(11):2573-81. Epub 2015 Jul 31.

Beta Cell Biochemistry Laboratory, John D. Dingell VA Medical Center, 4646 John R, Detroit, MI, 48201, USA.

Aims/hypothesis: Rho GTPases (Ras-related C3 botulinum toxin substrate 1 [Rac1] and cell division cycle 42 [Cdc42]) have been shown to regulate glucose-stimulated insulin secretion (GSIS) via cytoskeletal remodelling, trafficking and fusion of insulin-secretory granules with the plasma membrane. GTP loading of these G proteins, which is facilitated by GDP/GTP exchange factors, is a requisite step in the regulation of downstream effector proteins. Guanine nucleotide exchange factor VAV2 (VAV2), a member of the Dbl family of proteins, has been identified as one of the GDP/GTP exchange factors for Rac1. Despite recent evidence on the regulatory roles of VAV2 in different cell types, roles of this guanine nucleotide exchange factor in the signalling events leading to GSIS remain undefined. Using immunological, short interfering RNA (siRNA), pharmacological and microscopic approaches we investigated the role of VAV2 in GSIS from islet beta cells.

Methods: Co-localisation of Rac1 and VAV2 was determined by Triton X-114 phase partition and confocal microscopy. Glucose-induced actin remodelling was quantified by live cell imaging using the LifeAct-GFP fluorescent biosensor. Rac1 activation was determined by G protein linked immunosorbent assay (G-LISA).

Results: Western blotting indicated that VAV2 is expressed in INS-1 832/13 beta cells, normal rat islets and human islets. Vav2 siRNA markedly attenuated GSIS in INS-1 832/13 cells. Ehop-016, a newly discovered small molecule inhibitor of the VAV2-Rac1 interaction, or siRNA-mediated knockdown of VAV2 markedly attenuated glucose-induced Rac1 activation and GSIS in INS-1 832/13 cells. Pharmacological findings were recapitulated in primary rat islets. A high glucose concentration promoted co-localisation of Rac1 and VAV2. Real-time imaging in live cells indicated a significant inhibition of glucose-induced cortical actin remodelling by Ehop-016.

Conclusions/interpretation: Our data provide the first evidence to implicate VAV2 in glucose-induced Rac1 activation, actin remodelling and GSIS in pancreatic beta cells.
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http://dx.doi.org/10.1007/s00125-015-3707-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4591202PMC
November 2015

Protein prenylation in islet β-cell function in health and diabetes: Putting the pieces of the puzzle together.

Biochem Pharmacol 2015 Dec 26;98(3):363-70. Epub 2015 Jul 26.

Kresge Eye Institute, Wayne State University, Detroit, MI 48201, United States.

Post-translational prenylation involves incorporation of 15-(farnesyl) or 20-(geranylgeranyl) carbon derivatives of mevalonic acid into highly conserved C-terminal cysteines of proteins. The farnesyl transferase (FTase) and the geranylgeranyl transferase (GGTase) mediate incorporation of farnesyl and geranylgeranyl groups, respectively. At least 300 proteins are prenylated in the human genome; the majority of these are implicated in cellular processes including growth, differentiation, cytoskeletal function and vesicle trafficking. From a functional standpoint, isoprenylation is requisite for targeting of modified proteins to relevant cellular compartments for regulation of effector proteins. Pharmacological and molecular biological studies have provided compelling evidence for key roles of this signaling pathway in physiological insulin secretion in normal rodent and human islets. Recent evidence indicates that inhibition of prenylation results in mislocalization of unprenylated proteins, and surprisingly, they remain in active (GTP-bound) conformation. Sustained activation of G proteins has been reported in mice lacking GGTase, suggesting alternate mechanisms for the activation of non-prenylated G proteins. These findings further raise an interesting question if mislocalized, non-prenylated and functionally active G proteins cause cellular pathology since aberrant protein prenylation has been implicated in the onset of cardiovascular disease and diabetes. Herein, we overview the existing evidence to implicate prenylation in islet function and potential defects in this signaling pathways in the diabetic β-cell. We will also identify critical knowledge gaps that need to be addressed for the development of therapeutics to halt defects in these signaling steps in β cells in models of impaired insulin secretion, metabolic stress and diabetes.
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http://dx.doi.org/10.1016/j.bcp.2015.07.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6692653PMC
December 2015

COPII-Dependent ER Export: A Critical Component of Insulin Biogenesis and β-Cell ER Homeostasis.

Mol Endocrinol 2015 Aug 17;29(8):1156-69. Epub 2015 Jun 17.

Department of Physiology (J.F., B.P.J., F.S., J.W., T.B., X.C.) and Center for Molecular Medicine and Genetics (X.Z., K.Z.), School of Medicine, Department of Physics and Astronomy (T.S.), College of Liberal Arts and Sciences, and Department of Pharmaceutical Sciences (A.K.), Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, and John D. Dingell VA Medical Center (A.K.), Detroit, Michigan 48201; Department of Internal Medicine (M.L.), University of Michigan, Ann Arbor, Michigan 48109; and Department of Pathology (D.J.T.), Microscopy Imaging Center, University of Vermont College of Medicine, Burlington, Vermont 05405.

Pancreatic β-cells possess a highly active protein synthetic and export machinery in the endoplasmic reticulum (ER) to accommodate the massive production of proinsulin. ER homeostasis is vital for β-cell functions and is maintained by the delicate balance between protein synthesis, folding, export, and degradation. Disruption of ER homeostasis by diabetes-causing factors leads to β-cell death. Among the 4 components to maintain ER homeostasis in β-cells, the role of ER export in insulin biogenesis is the least understood. To address this knowledge gap, the present study investigated the molecular mechanism of proinsulin ER export in MIN6 cells and primary islets. Two inhibitory mutants of the secretion-associated RAS-related protein (Sar)1 small GTPase, known to specifically block coat protein complex II (COPII)-dependent ER export, were overexpressed in β-cells using recombinant adenoviruses. Results from this approach, as well as small interfering RNA-mediated Sar1 knockdown, demonstrated that defective Sar1 function blocked proinsulin ER export and abolished its conversion to mature insulin in MIN6 cells, isolated mouse, and human islets. It is further revealed, using an in vitro vesicle formation assay, that proinsulin was packaged into COPII vesicles in a GTP- and Sar1-dependent manner. Blockage of COPII-dependent ER exit by Sar1 mutants strongly induced ER morphology change, ER stress response, and β-cell apoptosis. These responses were mediated by the PKR (double-stranded RNA-dependent kinase)-like ER kinase (PERK)/eukaryotic translation initiation factor 2α (p-eIF2α) and inositol-requiring protein 1 (IRE1)/x-box binding protein 1 (Xbp1) pathways but not via activating transcription factor 6 (ATF6). Collectively, results from the study demonstrate that COPII-dependent ER export plays a vital role in insulin biogenesis, ER homeostasis, and β-cell survival.
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http://dx.doi.org/10.1210/me.2015-1012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4517999PMC
August 2015

Tiam1-Rac1 Axis Promotes Activation of p38 MAP Kinase in the Development of Diabetic Retinopathy: Evidence for a Requisite Role for Protein Palmitoylation.

Cell Physiol Biochem 2015 30;36(1):208-20. Epub 2015 Apr 30.

Ophthalmology, Wayne State University, Detroit, MI, USA.

Background/aims: Evidence in multiple tissues, including retina, suggests generation of reactive oxygen species (ROS) and the ensuing oxidative stress as triggers for mitochondrial defects and cell apoptosis. We recently reported novel roles for Tiam1-Rac1-Nox2 axis in retinal mitochondrial dysfunction and cell death leading to the development of diabetic retinopathy. Herein, we tested the hypothesis that activation of p38 MAP kinase, a stress kinase, represents the downstream signaling event to Rac1-Nox2 activation in diabetes-induced metabolic stress leading to capillary cell apoptosis.

Methods: Activation of p38 MAP kinase was quantified by Western blotting in retinal endothelial cells incubated with high glucose (20 mM) for up to 96 hours, a duration where mitochondrial dysfunction and capillary cell apoptosis can be observed. NSC23766 and 2-bromopalmitate (2-BP) were used to assess the roles of Tiam1-Rac1 and palmitoylation pathways, respectively.

Results: Activation of p38 MAP kinase was observed as early as 3 hours after high glucose exposure, and continued until 96 hours. Consistent with this, p38 MAP kinase activation was significantly higher in the retina from diabetic mice compared to age-matched normal mice. NSC23766 markedly attenuated hyperglycemia-induced activation of p38 MAP kinase. Lastly, 2-BP inhibited glucose-induced Rac1, Nox2 and p38 MAP kinase activation in endothelial cells.

Conclusions: Tiam1-Rac1-mediated activation of Nox2 and p38 MAP kinase constitutes early signaling events leading to mitochondrial dysfunction and the development of diabetic retinopathy. Our findings also provide the first evidence to implicate novel roles for protein palmitoylation in this signaling cascade.
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http://dx.doi.org/10.1159/000374065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4435616PMC
February 2016

Phagocyte-like NADPH oxidase (Nox2) promotes activation of p38MAPK in pancreatic β-cells under glucotoxic conditions: Evidence for a requisite role of Ras-related C3 botulinum toxin substrate 1 (Rac1).

Biochem Pharmacol 2015 Jun 14;95(4):301-10. Epub 2015 Apr 14.

Beta-Cell Biochemistry Laboratory, John D. Dingell VA Medical Center, and Department of Pharmaceutical Sciences, Wayne State University, Detroit, MI 48201, USA. Electronic address:

It is well established that glucotoxicity (caused by high glucose concentrations; HG) underlies pathogenesis of islet dysfunction in diabetes. We have recently demonstrated that Nox2 plays a requisite role in the generation of reactive oxygen species (ROS) under HG conditions, resulting in mitochondrial dysregulation and loss of islet β-cell function. Herein, we investigated roles of Nox2 in the regulation of downstream stress kinase (p38MAPK) activation under HG conditions (20mM; 24h) in normal rodent islets and INS-1 832/13 cells. We observed that gp91-ds-tat, a specific inhibitor of Nox2, but not its inactive analog, significantly attenuated HG-induced Nox2 activation, ROS generation and p38MAPK activation, thus suggesting that Nox2 activation couples with p38MAPK activation. Since Rac1, is an integral member of the Nox2 holoenzyme, we also assessed the effects of Rac1 inhibitors (EHT 1864, NSC23766 and Ehop-016) on HG-induced p38MAPK activation in isolated β-cells. We report a significant inhibition of p38MAPK phosphorylation by Rac1 inhibitors, implying a regulatory role for Rac1 in promoting the Nox2-p38MAPK signaling axis in the β-cell under the duress of HG. 2-Bromopalmitate, a known inhibitor of protein (Rac1) palmitoylation, significantly reduced HG-induced p38MAPK phosphorylation. However, GGTI-2147, a specific inhibitor of geranylgeranylation of Rac1, failed to exert any significant effects on HG-induced p38MAPK activation. In conclusion, we present the first evidence that the Rac1-Nox2 signaling module plays novel regulatory roles in HG-induced p38MAPK activation and loss in glucose-stimulated insulin secretion (GSIS) culminating in metabolic dysfunction and the onset of diabetes.
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http://dx.doi.org/10.1016/j.bcp.2015.04.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6684092PMC
June 2015

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

Cell Signal 2015 Jun 26;27(6):1159-67. Epub 2015 Feb 26.

John D. Dingell VA Medical Center, Detroit, MI 48201, USA; Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, Detroit, MI 48201, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.cellsig.2015.02.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4380787PMC
June 2015