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1.
Am J Physiol Regul Integr Comp Physiol ; 318(5): R1004-R1013, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32292063

RESUMO

Both reactive nitrogen and oxygen species (RNS and ROS), such as nitric oxide, peroxynitrite, and hydrogen peroxide, have been implicated as mediators of pancreatic ß-cell damage during the pathogenesis of autoimmune diabetes. While ß-cells are thought to be vulnerable to oxidative damage due to reportedly low levels of antioxidant enzymes, such as catalase and glutathione peroxidase, we have shown that they use thioredoxin reductase to detoxify hydrogen peroxide. Thioredoxin reductase is an enzyme that participates in the peroxiredoxin antioxidant cycle. Peroxiredoxins are expressed in ß-cells and, when overexpressed, protect against oxidative stress, but the endogenous roles of peroxiredoxins in the protection of ß-cells from oxidative damage are unclear. Here, using either glucose oxidase or menadione to continuously deliver hydrogen peroxide, or the combination of dipropylenetriamine NONOate and menadione to continuously deliver peroxynitrite, we tested the hypothesis that ß-cells use peroxiredoxins to detoxify both of these reactive species. Either pharmacological peroxiredoxin inhibition with conoidin A or specific depletion of cytoplasmic peroxiredoxin 1 (Prdx1) using siRNAs sensitizes INS 832/13 cells and rat islets to DNA damage and death induced by hydrogen peroxide or peroxynitrite. Interestingly, depletion of peroxiredoxin 2 (Prdx2) had no effect. Together, these results suggest that ß-cells use cytoplasmic Prdx1 as a primary defense mechanism against both ROS and RNS.


Assuntos
Dano ao DNA , Peróxido de Hidrogênio/toxicidade , Células Secretoras de Insulina/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Peroxirredoxinas/metabolismo , Ácido Peroxinitroso/toxicidade , Animais , Morte Celular , Linhagem Celular Tumoral , Citoplasma/enzimologia , Citoproteção , Inibidores Enzimáticos/farmacologia , Células Secretoras de Insulina/enzimologia , Células Secretoras de Insulina/patologia , Masculino , Peroxirredoxinas/antagonistas & inibidores , Peroxirredoxinas/genética , Quinoxalinas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos Sprague-Dawley , Transdução de Sinais , Tiorredoxina Redutase 1/metabolismo
2.
Mol Cell Biol ; 39(18)2019 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-31235477

RESUMO

In this report, we show that nitric oxide suppresses DNA damage response (DDR) signaling in the pancreatic ß-cell line INS 832/13 and rat islets by inhibiting intermediary metabolism. Nitric oxide is known to inhibit complex IV of the electron transport chain and aconitase of the Krebs cycle. Non-ß cells compensate by increasing glycolytic metabolism to maintain ATP levels; however, ß cells lack this metabolic flexibility, resulting in a nitric oxide-dependent decrease in ATP and NAD+ Like nitric oxide, mitochondrial toxins inhibit DDR signaling in ß cells by a mechanism that is associated with a decrease in ATP. Non-ß cells compensate for the effects of mitochondrial toxins with an adaptive shift to glycolytic ATP generation that allows for DDR signaling. Forcing non-ß cells to derive ATP via mitochondrial respiration (replacing glucose with galactose in the medium) and glucose deprivation sensitizes these cells to nitric oxide-mediated inhibition of DDR signaling. These findings indicate that metabolic flexibility is necessary to maintain DDR signaling under conditions in which mitochondrial oxidative metabolism is inhibited and support the inhibition of oxidative metabolism (decreased ATP) as one protective mechanism by which nitric oxide attenuates DDR-dependent ß-cell apoptosis.


Assuntos
Reparo do DNA/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Células Secretoras de Insulina/citologia , Óxido Nítrico/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular , Respiração Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA , Células Hep G2 , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Masculino , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , NAD/metabolismo , Ratos , Ratos Sprague-Dawley
3.
Stem Cells Transl Med ; 6(4): 1191-1201, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28224719

RESUMO

To address concerns regarding the tumorigenic potential of undifferentiated human pluripotent stem cells (hPSC) that may remain after in vitro differentiation and ultimately limit the broad use of hPSC-derivatives for therapeutics, we recently described a method to selectively eliminate tumorigenic hPSC from their progeny by inhibiting nicotinamide phosphoribosyltransferase (NAMPT). Limited exposure to NAMPT inhibitors selectively removes hPSC from hPSC-derived cardiomyocytes (hPSC-CM) and spares a wide range of differentiated cell types; yet, it remains unclear when and how cells acquire resistance to NAMPT inhibition during differentiation. In this study, we examined the effects of NAMPT inhibition among multiple time points of cardiomyocyte differentiation. Overall, these studies show that in vitro cardiomyogenic commitment and continued culturing provides resistance to NAMPT inhibition and cell survival is associated with the ability to maintain cellular ATP pools despite depletion of NAD levels. Unlike cells at earlier stages of differentiation, day 28 hPSC-CM can survive longer periods of NAMPT inhibition and maintain ATP generation by glycolysis and/or mitochondrial respiration. This is distinct from terminally differentiated fibroblasts, which maintain mitochondrial respiration during NAMPT inhibition. Overall, these results provide new mechanistic insight into how regulation of cellular NAD and energy pools change with hPSC-CM differentiation and further inform how NAMPT inhibition strategies could be implemented within the context of cardiomyocyte differentiation. Stem Cells Translational Medicine 2017;6:1191-1201.


Assuntos
Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Nicotinamida Fosforribosiltransferase/metabolismo , Acrilamidas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Humanos , Miócitos Cardíacos/metabolismo , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Piperidinas/farmacologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo
4.
Mol Cell Biol ; 36(15): 2067-77, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27185882

RESUMO

Nitric oxide, produced in pancreatic ß cells in response to proinflammatory cytokines, plays a dual role in the regulation of ß-cell fate. While nitric oxide induces cellular damage and impairs ß-cell function, it also promotes ß-cell survival through activation of protective pathways that promote ß-cell recovery. In this study, we identify a novel mechanism in which nitric oxide prevents ß-cell apoptosis by attenuating the DNA damage response (DDR). Nitric oxide suppresses activation of the DDR (as measured by γH2AX formation and the phosphorylation of KAP1 and p53) in response to multiple genotoxic agents, including camptothecin, H2O2, and nitric oxide itself, despite the presence of DNA damage. While camptothecin and H2O2 both induce DDR activation, nitric oxide suppresses only camptothecin-induced apoptosis and not H2O2-induced necrosis. The ability of nitric oxide to suppress the DDR appears to be selective for pancreatic ß cells, as nitric oxide fails to inhibit DDR signaling in macrophages, hepatocytes, and fibroblasts, three additional cell types examined. While originally described as the damaging agent responsible for cytokine-induced ß-cell death, these studies identify a novel role for nitric oxide as a protective molecule that promotes ß-cell survival by suppressing DDR signaling and attenuating DNA damage-induced apoptosis.


Assuntos
Camptotecina/farmacologia , Reparo do DNA/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Óxido Nítrico/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular , Dano ao DNA/efeitos dos fármacos , Células Hep G2 , Humanos , Células Secretoras de Insulina/citologia , Masculino , Camundongos , Especificidade de Órgãos , Fosforilação/efeitos dos fármacos , Células RAW 264.7 , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos
5.
Am J Physiol Regul Integr Comp Physiol ; 309(5): R525-34, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26084699

RESUMO

While insulinoma cells have been developed and proven to be extremely useful in studies focused on mechanisms controlling ß-cell function and viability, translating findings to human ß-cells has proven difficult because of the limited access to human islets and the absence of suitable insulinoma cell lines of human origin. Recently, a human ß-cell line, EndoC-ßH1, has been derived from human fetal pancreatic buds. The purpose of this study was to determine whether human EndoC-ßH1 cells respond to cytokines in a fashion comparable to human islets. Unlike most rodent-derived insulinoma cell lines that respond to cytokines in a manner consistent with rodent islets, EndoC-ßH1 cells fail to respond to a combination of cytokines (IL-1, IFN-γ, and TNF) in a manner consistent with human islets. Nitric oxide, produced following inducible nitric oxide synthase (iNOS) expression, is a major mediator of cytokine-induced human islet cell damage. We show that EndoC-ßH1 cells fail to express iNOS or produce nitric oxide in response to this combination of cytokines. Inhibitors of iNOS prevent cytokine-induced loss of human islet cell viability; however, they do not prevent cytokine-induced EndoC-ßH1 cell death. Stressed human islets or human islets expressing heat shock protein 70 (HSP70) are resistant to cytokines, and, much like stressed human islets, EndoC-ßH1 cells express HSP70 under basal conditions. Elevated basal expression of HSP70 in EndoC-ßH1 cells is consistent with the lack of iNOS expression in response to cytokine treatment. While expressing HSP70, EndoC-ßH1 cells fail to respond to endoplasmic reticulum stress activators, such as thapsigargin. These findings indicate that EndoC-ßH1 cells do not faithfully recapitulate the response of human islets to cytokines. Therefore, caution should be exercised when making conclusions regarding the actions of cytokines on human islets when using this human-derived insulinoma cell line.


Assuntos
Citocinas/farmacologia , Mediadores da Inflamação/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Insulinoma/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Neoplasias Pancreáticas/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/metabolismo , Metabolismo Energético/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Insulinoma/patologia , Interferon gama/farmacologia , Interleucina-1beta/farmacologia , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Masculino , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Neoplasias Pancreáticas/patologia , Fenótipo , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Técnicas de Cultura de Tecidos , Fator de Necrose Tumoral alfa/farmacologia
6.
Virology ; 483: 264-74, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26001649

RESUMO

Gammaherpesviruses are cancer-associated pathogens that establish life-long infection in most adults. Insufficiency of Ataxia-Telangiectasia mutated (ATM) kinase leads to a poor control of chronic gammaherpesvirus infection via an unknown mechanism that likely involves a suboptimal antiviral response. In contrast to the phenotype in the intact host, ATM facilitates gammaherpesvirus reactivation and replication in vitro. We hypothesized that ATM mediates both pro- and antiviral activities to regulate chronic gammaherpesvirus infection in an immunocompetent host. To test the proposed proviral activity of ATM in vivo, we generated mice with ATM deficiency limited to myeloid cells. Myeloid-specific ATM deficiency attenuated gammaherpesvirus infection during the establishment of viral latency. The results of our study uncover a proviral role of ATM in the context of gammaherpesvirus infection in vivo and support a model where ATM combines pro- and antiviral functions to facilitate both gammaherpesvirus-specific T cell immune response and viral reactivation in vivo.


Assuntos
Gammaherpesvirinae/fisiologia , Infecções por Herpesviridae/virologia , Células Mieloides/virologia , Ativação Viral , Adulto , Animais , Proteínas Mutadas de Ataxia Telangiectasia/deficiência , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Doença Crônica , Interações Hospedeiro-Patógeno , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout
7.
Stem Cells Transl Med ; 4(5): 483-93, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25834119

RESUMO

The tumorigenic potential of human pluripotent stem cells (hPSCs) is a major limitation to the widespread use of hPSC derivatives in the clinic. Here, we demonstrate that the small molecule STF-31 is effective at eliminating undifferentiated hPSCs across a broad range of cell culture conditions with important advantages over previously described methods that target metabolic processes. Although STF-31 was originally described as an inhibitor of glucose transporter 1, these data support the reclassification of STF-31 as a specific NAD⁺ salvage pathway inhibitor through the inhibition of nicotinamide phosphoribosyltransferase (NAMPT). These findings demonstrate the importance of an NAD⁺ salvage pathway in hPSC biology and describe how inhibition of NAMPT can effectively eliminate hPSCs from culture. These results will advance and accelerate the development of safe, clinically relevant hPSC-derived cell-based therapies.


Assuntos
Diferenciação Celular/efeitos dos fármacos , NAD/antagonistas & inibidores , Células-Tronco Pluripotentes/efeitos dos fármacos , Piridinas/farmacologia , Técnicas de Cultura de Células , Citocinas/antagonistas & inibidores , Humanos , NAD/metabolismo , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Células-Tronco Pluripotentes/citologia , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia
9.
J Biol Chem ; 289(16): 11454-11464, 2014 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-24610783

RESUMO

In this study, the effects of cytokines on the activation of the DNA double strand break repair factors histone H2AX (H2AX) and ataxia telangiectasia mutated (ATM) were examined in pancreatic ß cells. We show that cytokines stimulate H2AX phosphorylation (γH2AX formation) in rat islets and insulinoma cells in a nitric oxide- and ATM-dependent manner. In contrast to the well documented role of ATM in DNA repair, ATM does not appear to participate in the repair of nitric oxide-induced DNA damage. Instead, nitric oxide-induced γH2AX formation correlates temporally with the onset of irreversible DNA damage and the induction of apoptosis. Furthermore, inhibition of ATM attenuates cytokine-induced caspase activation. These findings show that the formation of DNA double strand breaks correlates with ATM activation, irreversible DNA damage, and ATM-dependent induction of apoptosis in cytokine-treated ß cells.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Células Secretoras de Insulina/metabolismo , Óxido Nítrico/metabolismo , Animais , Apoptose/fisiologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Caspases/genética , Caspases/metabolismo , Linhagem Celular Tumoral , Citocinas/genética , Citocinas/metabolismo , Quebras de DNA de Cadeia Dupla , Ativação Enzimática/fisiologia , Histonas , Células Secretoras de Insulina/citologia , Masculino , Óxido Nítrico/genética , Fosfoproteínas , Fosforilação/fisiologia , Ratos , Ratos Sprague-Dawley
10.
Free Radic Biol Med ; 69: 229-38, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24486553

RESUMO

Energy substrates metabolized through mitochondria (e.g., pyruvate, glutamine) are required for biosynthesis of macromolecules in proliferating cells. Because several mitochondrial proteins are known to be targets of S-nitrosation, we determined whether bioenergetics are modulated by S-nitrosation and defined the subsequent effects on proliferation. The nitrosating agent S-nitroso-L-cysteine (L-CysNO) was used to initiate intracellular S-nitrosation, and treatment decreased mitochondrial function and inhibited proliferation of MCF7 mammary adenocarcinoma cells. Surprisingly, the d-isomer of CysNO (D-CysNO), which is not transported into cells, also caused mitochondrial dysfunction and limited proliferation. Both L- and D-CysNO also inhibited cellular pyruvate uptake and caused S-nitrosation of thiol groups on monocarboxylate transporter 1, a proton-linked pyruvate transporter. These data demonstrate the importance of mitochondrial metabolism in proliferative responses in breast cancer and highlight a novel role for inhibition of metabolic substrate uptake through S-nitrosation of exofacial protein thiols in cellular responses to nitrosative stress.


Assuntos
Mitocôndrias/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Nitrosação , Ácido Pirúvico/metabolismo , Simportadores/metabolismo , Proliferação de Células/efeitos dos fármacos , Respiração Celular/efeitos dos fármacos , Respiração Celular/genética , Cisteína/administração & dosagem , Cisteína/análogos & derivados , Humanos , Células MCF-7 , Mitocôndrias/efeitos dos fármacos , Óxido Nítrico/metabolismo , S-Nitrosotióis/administração & dosagem , Compostos de Sulfidrila/metabolismo
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