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Dive into the research topics where Rajakrishnan Veluthakal is active.

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Featured researches published by Rajakrishnan Veluthakal.


Apoptosis | 2005

Ceramide induces mitochondrial abnormalities in insulin-secreting INS-1 cells: Potential mechanisms underlying ceramide-mediated metabolic dysfunction of the β cell

Rajakrishnan Veluthakal; R. Palanivel; Y. Zhao; P. McDonald; S. Gruber; Anjaneyulu Kowluru

C2-ceramide, a cell permeable analogue of ceramide [CER] markedly reduced mitochondrial membrane potential [MMP] in insulin-secreting INS cells, which was followed by a significant accumulation of cytochrome c [Cyt c] into the cytosolic compartment. In a manner akin to CER, exposure of these cells to interleukin-1β [IL-1β] also resulted in reduction in MMP and cytosolic accumulation of Cyt c. Further, long-term exposure of these cells to either CER [but not its inactive analogue] or IL-1β caused a marked reduction in their metabolic viability. However, unlike IL-1β, which increased nitric oxide [NO] release, CER-treatment of INS cells had no effects of CER on NO release were demonstrable. Together, these findings suggest that CER-induced mitochondrial effects may not be mediated via iNOS gene expression and NO production. CER also activated an okadaic acid -sensitive protein phosphatase [CAPP] in the purified mitochondrial fraction, suggesting that CAPP might represent one of the target proteins for CER in the β cell mitochondria. Together, our findings suggest direct detrimental effects of CER on mitochondrial function in β cells leading to their dysfunction and demise via apoptosis. Moreover, our findings provide evidence for a potential difference in the mechanisms underlying CER- and IL-1β-induced mitochondrial defects and apoptotic demise of the effete β cell.


Diabetes | 2010

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

Anjaneyulu Kowluru; Rajakrishnan Veluthakal; Christopher J. Rhodes; Vasudeva Kamath; Ismail Syed; Brandon J. Koch

OBJECTIVE Posttranslational prenylation (e.g., farnesylation) of small G-proteins is felt to be requisite for cytoskeletal remodeling and fusion of secretory vesicles with the plasma membrane. Here, we investigated roles of protein farnesylation in the signaling steps involved in Raf-1/extracellular signal–related kinase (ERK1/2) signaling pathway in glucose-induced Rac1 activation and insulin secretion in the pancreatic β-cell. RESEARCH DESIGN AND METHODS These studies were carried out in INS 832/13 cells and normal rat islets. Molecular biological (e.g., overexpression or small interfering RNA [siRNA]–mediated knockdown) and pharmacologic approaches were used to determine roles for farnesylation in glucose-mediated activation of ERK1/2, Rac1, and insulin secretion. Activation of ERK1/2 was determined by Western blotting. Rac1 activation (i.e., Rac1.GTP) was quantitated by p21-activated kinase pull-down assay. Insulin release was quantitated by enzyme-linked immunosorbent assay. RESULTS Coprovision of structure-specific inhibitors of farnesyl transferase (FTase; e.g., FTI-277 or FTI-2628) or siRNA-mediated knockdown of FTase β-subunit resulted in a significant inhibition of glucose-stimulated ERK1/2 and Rac1 activation and insulin secretion. Pharmacologic inhibition of Raf-1 kinase using GW-5074 markedly reduced the stimulatory effects of glucose on ERK1/2 phosphorylation, Rac1 activation, and insulin secretion, suggesting that Raf-1 kinase activation may be upstream to ERK1/2 and Rac1 activation leading to glucose-induced insulin release. Lastly, siRNA-mediated silencing of endogenous expression of ERK1/2 markedly attenuated glucose-induced Rac1 activation and insulin secretion. CONCLUSIONS Together, our findings provide the first evidence of a role for protein farnesylation in glucose-mediated regulation of the Raf/ERK signaling pathway culminating in the activation of Rac1, which has been shown to be necessary for cytoskeletal reorganization and exocytotic secretion of insulin.


Diabetes | 2007

Dominant-Negative α-Subunit of Farnesyl- and Geranyltransferase Inhibits Glucose-Stimulated, but Not KCl-Stimulated, Insulin Secretion in INS 832/13 Cells

Rajakrishnan Veluthakal; Hitchintan Kaur; Marc L. Goalstone; Anjaneyulu Kowluru

The majority of small G-proteins undergo posttranslational modifications (e.g., isoprenylation) at their C-terminal cysteine residues. Such modifications increase their hydrophobicity, culminating in translocation of the modified proteins to their relevant membranous sites for interaction with their respective effectors. Previously, we reported glucose-dependent activation and membrane association of Rac1 in INS 832/13 cells. We also demonstrated modulatory roles for Rac1/GDP dissociation inhibitor in glucose-stimulated insulin secretion (GSIS) in INS 832/13 cells, further affirming roles for Rac1 in GSIS. Herein, we demonstrate that geranylgeranyltransferase inhibitor-2147 (GGTI-2147), an inhibitor of protein prenylation, markedly increased cytosolic accumulation of Rac1 and elicited significant inhibition of GSIS from INS 832/13 cells. In the current study, we also examined the localization of protein prenyltransferases (PPTases) and regulation of GSIS by PPTases in INS 832/13 cells. Western blot analyses indicated that the regulatory α-subunit and the structural β-subunit of PPTase holoenzyme are predominantly cytosolic in their distribution. Overexpression of an inactive mutant of the regulatory α-subunit of PPTase markedly attenuated glucose- but not KCl-induced insulin secretion from INS 832/13 cells. Together, our findings provide the first evidence for the regulation of GSIS by PPTase in INS 832/13 cells. Furthermore, they support our original hypothesis that prenylation of specific G-proteins may be necessary for GSIS.


Biochemical Pharmacology | 2009

Regulatory roles for Tiam1, a guanine nucleotide exchange factor for Rac1, in glucose-stimulated insulin secretion in pancreatic β-cells

Rajakrishnan Veluthakal; Suresh Vasu Madathilparambil; Phillip McDonald; Lawrence Karl Olson; Anjaneyulu Kowluru

Using various biochemical, pharmacological and molecular biological approaches, we have recently reported regulatory roles for Rac1, a small G-protein, in glucose-stimulated insulin secretion (GSIS). However, little is understood with respect to localization of, and regulation by, specific regulatory factors of Rac1 in GSIS. Herein, we investigated regulatory roles for Tiam1, a specific nucleotide exchange factor (GEF) for Rac1, in GSIS in pancreatic beta-cells. Western blot analysis indicated that Tiam1 is predominantly cytosolic in distribution. NSC23766, a specific inhibitor of Tiam1-mediated activation of Rac1, markedly attenuated glucose-induced, but not KCl-induced insulin secretion in INS 832/13 cells and normal rat islets. Further, NSC23766 significantly reduced glucose-induced activation (i.e. GTP-bound form) and membrane association of Rac1 in INS 832/13 cells and rat islets. Moreover, siRNA-mediated knock-down of Tiam1 markedly inhibited glucose-induced membrane trafficking and activation of Rac1 in INS 832/13 cells. Interestingly, however, in contrast to the inhibitory effects of NSC23766, Tiam1 gene depletion potentiated GSIS in these cells; such a potentiation of GSIS was sensitive to extracellular calcium. Together, our studies present the first evidence for a regulatory role for Tiam1/Rac1-sensitive signaling step in GSIS. They also provide evidence for the existence of a potential Rac1/Tiam1-independent, but calcium-sensitive component for GSIS in these cells.


Diabetologia | 2014

TIAM1-RAC1 signalling axis-mediated activation of NADPH oxidase-2 initiates mitochondrial damage in the development of diabetic retinopathy.

Renu A. Kowluru; Anjaneyulu Kowluru; Rajakrishnan Veluthakal; Ghulam Mohammad; Ismail Syed; Julia M. Santos; Manish Mishra

Aims/hypothesisIn diabetes, increased retinal oxidative stress is seen before the mitochondria are damaged. Phagocyte-like NADPH oxidase-2 (NOX2) is the predominant cytosolic source of reactive oxygen species (ROS). Activation of Ras-related C3 botulinum toxin substrate 1 (RAC1), a NOX2 holoenzyme member, is necessary for NOX2 activation and ROS generation. In this study we assessed the role of T cell lymphoma invasion and metastasis (TIAM1), a guanine nucleotide exchange factor for RAC1, in RAC1 and NOX2 activation and the onset of mitochondrial dysfunction in in vitro and in vivo models of glucotoxicity and diabetes.MethodsRAC1 and NOX2 activation, ROS generation, mitochondrial damage and cell apoptosis were quantified in bovine retinal endothelial cells exposed to high glucose concentrations, in the retina from normal and streptozotocin-induced diabetic rats and mice, and the retina from human donors with diabetic retinopathy.ResultsHigh glucose activated RAC1 and NOX2 (expression and activity) and increased ROS in endothelial cells before increasing mitochondrial ROS and mitochondrial DNA (mtDNA) damage. N6-[2-[[4-(diethylamino)-1-methylbutyl]amino]-6-methyl-4-pyrimidinyl]-2-methyl-4,6-quinolinediamine, trihydrochloride (NSC23766), a known inhibitor of TIAM1–RAC1, markedly attenuated RAC1 activation, total and mitochondrial ROS, mtDNA damage and cell apoptosis. An increase in NOX2 expression and membrane association of RAC1 and p47phox were also seen in diabetic rat retina. Administration of NSC23766 to diabetic mice attenuated retinal RAC1 activation and ROS generation. RAC1 activation and p47phox expression were also increased in the retinal microvasculature from human donors with diabetic retinopathy.Conclusions/interpretationThe TIAM1–RAC1–NOX2 signalling axis is activated in the initial stages of diabetes to increase intracellular ROS leading to mitochondrial damage and accelerated capillary cell apoptosis. Strategies targeting TIAM1–RAC1 signalling could have the potential to halt the progression of diabetic retinopathy in the early stages of the disease.


Journal of Cellular and Molecular Medicine | 2009

A novel histone deacetylase inhibitor prevents IL‐1β induced metabolic dysfunction in pancreatic β‐cells

Laura Susick; Thulani Senanayake; Rajakrishnan Veluthakal; Patrick M. Woster; Anjaneyulu Kowluru

The histone deacetylase (HDAC) inhibitor trichostatin A (TSA) has recently been shown to inhibit deleterious effects of cytokines on β‐cells, but it is unable to protect β‐cells from death due to its own cytotoxicity. Herein, we investigated novel HDAC inhibitors for their cytoprotective effects against IL‐1β‐induced damage to isolated β‐cells. We report that three novel compounds (THS‐73–44, THS‐72–5 and THS‐78–5) significantly inhibited HDAC activity and increased the acetylation of histone H4 in isolated β‐cells. Further, these compounds exerted no toxic effects on metabolic cell viability in these cells. However, among the three compounds tested, only THS‐78–5 protected against IL‐1β‐mediated loss in β‐cell viability. THS‐78–5 was also able to attenuate IL‐1β‐induced inducible nitric oxide synthase expression and subsequent NO release. Our data also indicate that the cytoprotective properties of THS‐78–5 against IL‐1β‐mediated effects may, in part, be due to inhibition of IL‐1β‐induced transactivation of nuclear factor κB (NF‐κB) in these cells. Together, we provide evidence for a novel HDAC inhibitor with a significant potential to prevent IL‐1β‐mediated effects on isolated β‐cells. Potential implications of these findings in the development of novel therapeutics to prevent deleterious effects of cytokines and the onset of autoimmune diabetes are discussed.


Cellular Physiology and Biochemistry | 2015

Tiam1-rac1 axis promotes activation of p38 MAP kinase in the development of diabetic retinopathy: Evidence for a requisite role for protein palmitoylation

Rajakrishnan Veluthakal; Binit Kumar; Ghulam Mohammad; Anjaneyulu Kowluru; Renu A. Kowluru

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.


Biochemical Pharmacology | 2015

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).

Vaibhav Sidarala; Rajakrishnan Veluthakal; Khadija Syeda; Cornelis Vlaar; Philip Newsholme; Anjaneyulu Kowluru

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.


Molecular and Cellular Biochemistry | 2009

Down-regulation of expression and function of nucleoside diphosphate kinase in insulin-secreting β-cells under in vitro conditions of glucolipotoxicity

Rajakrishnan Veluthakal; Madathilparambil V. Suresh; Anjaneyulu Kowluru

Previously, we reported a significant reduction in expression and the activity of nucleoside diphosphate kinase (NDP kinase) in islets derived from the Goto-Kakizaki rat (GK rat), an animal model for type 2 diabetes. Herein, we examined the effects of chronic exposure of insulin-secreting β-(INS 832/13) cells to high glucose (a model for glucotoxicity), palmitate (a model for lipotoxicity), or glucose plus palmitate (a model for glucolipotoxicity) on the expression and activity of nm23-H1 (NDP kinase A) and nm23-H2 (NDP kinase B). Our findings indicate a marked reduction in the expression of both nm23-H1 and nm23-H2 and the associated NDP kinase activity under each of these conditions. A cell-permeable analog of ceramide (CER) also mimicked the effects of palmitate in significantly reducing the expression of nm23-H1 and nm23-H2 and NDP kinase activity in these cells. These findings suggest that de novo generation of intracellular CER from palmitate might represent at least one of the signaling steps involved in lipid-induced effects on NDP kinase expression and function in β-cells. Based on these data, we conclude that glucolipotoxic conditions significantly impair expression and function of NDP kinase in pancreatic β-cells. Potential significance of these findings, specifically at the level of abnormal G-protein activation and impaired insulin secretion under glucolipotoxic conditions is discussed.


Journal of Cellular and Molecular Medicine | 2008

Regulatory roles for histone deacetylation in IL-1β-induced nitric oxide release in pancreatic β-cells

Laura L. Susick; Rajakrishnan Veluthakal; M. V. Suresh; T. Hadden; Anjaneyulu Kowluru

Histone (de)acetylases control gene transcription via modification of the chromatin structure. Herein, we investigated potential roles for histone deacetylation (or hypoacetylation) in interleukin‐1β (IL‐1β)‐mediated inducible nitric oxide synthase (iNOS) and nitric oxide (NO) release in insulin‐secreting INS 832/13 (INS) cells. Western blot analysis suggested localization of members of Class 1 and Class 2 families of histone deacetylases (HDACs) in these cells. Trichostatin A (TSA), a known inhibitor of HDACs, markedly reduced IL‐1β‐mediated iNOS expression and NO release from these cells in a concentration‐dependent manner. TSA also promoted hyperacetylation of histone H4 under conditions in which it inhibited IL‐1β‐mediated effects on isolated β cells. Rottlerin, a known inhibitor of protein kinase Cδ, also increased histone H4 acetylation, and inhibited IL‐1β‐induced iNOS expression and NO release in these cells. It appears that the putative mechanism underlying the stimulatory effects of rottlerin on acetylation status of histone H4 are distinct from the HDAC inhibitory property of TSA, since rottlerin failed to inhibit HDAC activity in nuclear extracts isolated from INS cells. These data are suggestive of potential regulatory effects of rottlerin at the level of increasing the histone acetyltransferase activity in these cells. Together our studies present the first evidence to suggest a PKCδ‐mediated signalling step, which promotes hypoacetylation of candidate histones culminating in IL‐1β‐induced metabolic dysfunction of the isolated β cell.

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Ismail Syed

Wayne State University

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Rajesh Amin

Wayne State University

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Cornelis Vlaar

University of Puerto Rico

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